Cationic neurotoxins

ABSTRACT

Engineered clostridial toxins comprising an amino acid modification that increases the isoelectric point of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the isoelectric point of an otherwise identical clostridial toxin lacking the modification, wherein the modification is not located in the clostridial toxin binding domain (H C  domain).

The present invention relates to engineered clostridial toxinscomprising at least one amino acid modification, and the use of suchengineered clostridial toxins in medicine and therapy.

Bacteria in the genus Clostridia produce highly potent and specificprotein toxins, which can poison neurons and other cells to which theyare delivered. Examples of such clostridial toxins include theneurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT)serotypes A-G, as well as those produced by C. baratii and C. butyricum.

Among the clostridial toxins are some of the most potent toxins known.By way of example, botulinum neurotoxins have median lethal dose (LD₅₀)values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype.Both tetanus and botulinum toxins act by inhibiting the function ofaffected neurons, specifically the release of neurotransmitters. Whilebotulinum toxin acts at the neuromuscular junction and inhibitscholinergic transmission in the peripheral nervous system, tetanus toxinacts in the central nervous system.

In nature, clostridial toxins are synthesised as a single-chainpolypeptide that is modified post-translationally by a proteolyticcleavage event to form two polypeptide chains joined together by adisulphide bond. Cleavage occurs at a specific cleavage site, oftenreferred to as the activation site, that is located between the cysteineresidues that provide the inter-chain disulphide bond. It is thisdi-chain form that is the active form of the toxin. The two chains aretermed the heavy chain (H-chain), which has a molecular mass ofapproximately 100 kDa, and the light chain (L-chain), which has amolecular mass of approximately 50 kDa. The H-chain comprises anN-terminal translocation component (H_(N) domain) and a C-terminaltargeting component (H_(C) domain). The cleavage site is located betweenthe L-chain and the translocation domain components. Following bindingof the H_(C) domain to its target neuron and internalisation of thebound toxin into the cell via an endosome, the H_(N) domain translocatesthe L-chain across the endosomal membrane and into the cytosol, and theL-chain provides a protease function (also known as a non-cytotoxicprotease).

Non-cytotoxic proteases act by proteolytically cleaving intracellulartransport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, orSyntaxin)—see Gerald K (2002) “Cell and Molecular Biology” (4th edition)John Wiley & Sons, Inc. The acronym SNARE derives from the term SolubleNSF Attachment Receptor, where NSF means N-ethylmaleimide-SensitiveFactor. SNARE proteins are integral to intracellular vesicle fusion, andthus to secretion of molecules via vesicle transport from a cell. Theprotease function is a zinc-dependent endopeptidase activity andexhibits a high substrate specificity for SNARE proteins. Accordingly,once delivered to a desired target cell, the non-cytotoxic protease iscapable of inhibiting cellular secretion from the target cell. TheL-chain proteases of clostridial toxins are non-cytotoxic proteases thatcleave SNARE proteins.

In view of the ubiquitous nature of SNARE proteins, clostridial toxinssuch as botulinum toxin have been successfully employed in a wide rangeof therapies.

By way of example, we refer to William J. Lipham, Cosmetic and ClinicalApplications of Botulinum Toxin (Slack, Inc., 2004), which describes theuse of clostridial toxins, such as botulinum neurotoxins (BoNTs),BoNT/A, BoNT/B, BoNT/C₁, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and tetanusneurotoxin (TeNT), to inhibit neuronal transmission in a number oftherapeutic and cosmetic or aesthetic applications—for example, marketedbotulinum toxin products are currently approved as therapeutics forindications including focal spasticity, upper limb spasticity, lowerlimb spasticity, cervical dystonia, blepharospasm, hemifacial spasm,hyperhidrosis of the axillae, chronic migraine, neurogenic detrusoroveractivity, glabellar lines, and severe lateral canthal lines. Inaddition, clostridial toxin therapies are described for treatingneuromuscular disorders (see U.S. Pat. No. 6,872,397); for treatinguterine disorders (see US 2004/0175399); for treating ulcers andgastroesophageal reflux disease (see US 2004/0086531); for treatingdystonia (see U.S. Pat. No. 6,319,505); for treating eye disorders (seeUS 2004/0234532); for treating blepharospasm (see US 2004/0151740); fortreating strabismus (see US 2004/0126396); for treating pain (see U.S.Pat. No. 6,869,610, U.S. Pat. No. 6,641,820, U.S. Pat. No. 6,464,986,and U.S. Pat. No. 6,113,915); for treating fibromyalgia (see U.S. Pat.No. 6,623,742, US 2004/0062776); for treating lower back pain (see US2004/0037852); for treating muscle injuries (see U.S. Pat. No.6,423,319); for treating sinus headache (see U.S. Pat. No. 6,838,434);for treating tension headache (see U.S. Pat. No. 6,776,992); fortreating headache (see U.S. Pat. No. 6,458,365); for reduction ofmigraine headache pain (see U.S. Pat. No. 5,714,469); for treatingcardiovascular diseases (see U.S. Pat. No. 6,767,544); for treatingneurological disorders such as Parkinson's disease (see U.S. Pat. No.6,620,415, U.S. Pat. No. 6,306,403); for treating neuropsychiatricdisorders (see US 2004/0180061, US 2003/0211121); for treating endocrinedisorders (see U.S. Pat. No. 6,827,931); for treating thyroid disorders(see U.S. Pat. No. 6,740,321); for treating cholinergic influenced sweatgland disorders (see U.S. Pat. No. 6,683,049); for treating diabetes(see U.S. Pat. No. 6,337,075, U.S. Pat. No. 6,416,765); for treating apancreatic disorder (see U.S. Pat. No. 6,261,572, U.S. Pat. No.6,143,306); for treating cancers such as bone tumors (see U.S. Pat. No.6,565,870, U.S. Pat. No. 6,368,605, U.S. Pat. No. 6,139,845, US2005/0031648); for treating otic disorders (see U.S. Pat. No. 6,358,926,U.S. Pat. No. 6,265,379); for treating autonomic disorders such asgastrointestinal muscle disorders and other smooth muscle dysfunction(see U.S. Pat. No. 5,437,291); for treatment of skin lesions associatedwith cutaneous cell-proliferative disorders (see U.S. Pat. No.5,670,484); for management of neurogenic inflammatory disorders (seeU.S. Pat. No. 6,063,768); for reducing hair loss and stimulating hairgrowth (see U.S. Pat. No. 6,299,893); for treating downturned mouth (seeU.S. Pat. No. 6,358,917); for reducing appetite (see US 2004/40253274);for dental therapies and procedures (see US 2004/0115139); for treatingneuromuscular disorders and conditions (see US 2002/0010138); fortreating various disorders and conditions and associated pain (see US2004/0013692); for treating conditions resulting from mucushypersecretion such as asthma and COPD (see WO 00/10598); and fortreating non-neuronal conditions such as inflammation, endocrineconditions, exocrine conditions, immunological conditions,cardiovascular conditions, bone conditions (see WO 01/21213). All of theabove publications are hereby incorporated by reference in theirentirety.

The use of non-cytotoxic proteases such as clostridial toxins (e.g.BoNTs and TeNT) in therapeutic and cosmetic treatments of humans andother mammals is anticipated to expand to an ever-widening range ofdiseases and ailments that can benefit from the properties of thesetoxins.

To avoid systemic neurological effects, many clostridial toxin therapiesutilise direct administration of the clostridial toxin therapeutic to agiven target site (such as a target tissue). A problem whenadministering clostridial toxin-based therapeutics in this fashion isthe spread of toxin away from the administration site and intosurrounding tissue or systemic circulation. The diffusion of toxin awayfrom the target tissue is believed to be responsible for undesirableside effects that in extreme cases may be life threatening. This can bea particular concern when using clostridial toxin therapeutics (such asBoNT therapeutics) at high doses, concentrations and injection volumes.Adverse effects associated with this problem that have been reported forcommercial BoNT/A therapeutics include asthenia, generalised muscleweakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinaryincontinence, and breathing difficulties. Swallowing and breathingdifficulties can be life threatening and there have been reported deathsrelated to the spread of toxin effects.

There is therefore a need in the art for clostridial toxins which haveproperties of increased tissue retention at the site of administration,and which accordingly exhibit a reduction in diffusion away from theadministration site, as compared to known clostridial toxins.

The present invention solves the above problem by providing engineeredclostridial toxins, as specified in the claims.

In one aspect, the invention provides an engineered clostridial toxincomprising at least one (for example, at least one, two or three) aminoacid modification, wherein said at least one amino acid modificationincreases the isoelectric point (pI) of the engineered clostridial toxinto a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical clostridial toxin lacking said at least one aminoacid modification, and wherein said at least one amino acid modificationis not located in the clostridial toxin binding domain (H_(C) domain).

In one embodiment, “not located in the clostridial toxin binding domain(H_(C)) domain” means that said at least one amino acid modification islocated in the clostridial toxin H_(N) domain or in the clostridialtoxin light chain.

In one embodiment, the invention provides an engineered clostridialtoxin comprising at least one (for example, at least one, two or three)amino acid modification, wherein said at least one amino acidmodification increases the isoelectric point (pI) of the engineeredclostridial toxin to a value that is at least 0.2 (for example, at least0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pIof an otherwise identical clostridial toxin lacking said at least oneamino acid modification, and wherein said at least one amino acidmodification is located in the clostridial toxin translocation domain(H_(N) domain).

In another embodiment, the invention provides an engineered clostridialtoxin comprising at least one (for example, at least one, two or three)amino acid modification, wherein said at least one amino acidmodification increases the isoelectric point (pI) of the engineeredclostridial toxin to a value that is at least 0.2 (for example, at least0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pIof an otherwise identical clostridial toxin lacking said at least oneamino acid modification, and wherein said at least one amino acidmodification is located in the clostridial toxin light chain.

In one embodiment, wherein said at least one amino acid modification islocated in the clostridial toxin light chain, said at least one aminoacid modification does not introduce into the clostridial toxin lightchain an E3 ligase recognition motif. Thus, in one embodiment, the lightchain of an engineered clostridial toxin of the invention does notcomprise an E3 ligase recognition motif.

As used above, the term “E3 ligase recognition motif” refers to amodification of the light chain that results in accelerated degradationof the neurotoxin polypeptide by endogenous proteasome degradationpathways present in a subject to which the neurotoxin has been applied.An “E3 ligase recognition” motif is a structural motif that allowsrecognition of the motif and binding to the motif by an E3 ligase (alsoknown as an E3 ubiquitin ligase; thus, an “E3 ligase recognition motif”may also be referred to as an “E3 ubiquitin ligase recognition motif”).E3 ligase recognition motifs will be familiar to a person skilled in theart.

Examples of E3 ligase recognition motifs include the following sequences(wherein “X” may represent any of the naturally occurring amino acids):

E3 ubiquitin ligase Recognition motif (consensus) VBCCul2ALAPYIP (SEQ ID NO: 9) MDM2 XXFXXWXXLXX (SEQ ID NO: 10) MNM2RFMDYWEGL (SEQ ID NO: 11) FXXXLWXXL (SEQ ID NO: 12) Smurf2ELESPPPPYSRYPM (SEQ ID NO: 13) RN181 KVGFFKR (SEQ ID NO: 14) E3alphaLLVRGRTLVV (SEQ ID NO: 15) SCF DRHDSGLDSM (SEQ ID NO: 16) SiahPXAXVXP (SEQ ID NO: 17) Itch PPXYXXM (SEQ ID NO: 18) Nedd4-2PPXY (SEQ ID NO: 19)

Further examples of E3 ligase recognition motifs include: ETFSDLWKLLPE(SEQ ID NO:20), TSFAEYWNLLSP (SEQ ID NO: 21), LTFEHYWAQLTS (SEQ ID NO:22), LTFEHWWAQLTS (SEQ ID NO: 23), LTFEHSWAQLTS (SEQ ID NO: 24),ETFEHNWAQLTS (SEQ ID NO: 25), LTFEHNWAQLTS (SEQ ID NO: 26), LTFEHWWASLTS(SEQ ID NO: 27), LTFEHWWSSLTS (SEQ ID NO: 28), LTFTHWWAQLTS (SEQ ID NO:29), ETFEHWWAQLTS (SEQ ID NO: 30), LTFEHWWSQLTS (SEQ ID NO: 31),LTFEHWWAQLLS (SEQ ID NO: 32), ETFEHWWSQLLS (SEQ ID NO: 33), RFMDYWEGL(SEQ ID NO: 34), MPRFMDYWEGLN (SEQ ID NO: 35), SQETFSDLWKLLPEN (SEQ IDNO: 36), and LTFEHNWAQLEN (SEQ ID NO: 37).

In one embodiment, wherein said at least one amino acid modification islocated in the clostridial toxin light chain, said at least one aminoacid modification does not introduce into the clostridial toxin lightchain an MDM2 E3 ligase recognition motif. Thus, in one embodiment, thelight chain of an engineered clostridial toxin of the invention does notcomprise an MDM2 E3 ligase recognition motif.

In one embodiment, wherein said at least one amino acid modification islocated in the clostridial toxin light chain, the engineered clostridialtoxin does not comprise an amino acid modification at an N-terminalproline.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,and wherein said at least one amino acid modification is located in theclostridial toxin light chain, said engineered BoNT/E does not comprisea substitution with lysine at any one of the following amino acidpositions: Q53, N72, N378, N379, R394, T400.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,and wherein said at least one amino acid modification is located in theclostridial toxin light chain, said engineered BoNT/E does not comprisea substitution with lysine at any one of the following amino acidpositions: Q53, N72, N378, N379, T400.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,and wherein said at least one amino acid modification is located in theclostridial toxin light chain, said engineered BoNT/E does not comprisea substitution with lysine at any three of the following amino acidpositions: Q53, N72, N378, N379, R394, T400.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,and wherein said at least one amino acid modification is located in theclostridial toxin light chain, said engineered BoNT/E does not comprisea substitution with lysine at any three of the following amino acidpositions: Q53, N72, N378, N379, T400.

In one embodiment, optionally wherein the at least one amino acidmodification is located in the clostridial toxin light chain, theengineered clostridial toxin is not a BoNT/E.

The engineered clostridial toxins of the invention do not comprise anyamino acid modifications located in the clostridial toxin H_(C) domain.Thus, in an engineered clostridial toxin of the invention, said at leastone amino acid modification is not located in the clostridial toxinH_(C) domain.

In one embodiment, wherein said at least one amino acid modification islocated in the clostridial toxin light chain, said at least one aminoacid modification does not comprise the substitution of an amino acidresidue with a lysine residue.

In one embodiment, wherein the engineered clostridial toxin is anengineered clostridial toxin as described above, said at least one aminoacid modification comprises substitution of an acidic amino acid residueor an uncharged amino acid residue with a lysine or arginine residue.

In one embodiment, wherein the engineered clostridial toxin is anengineered clostridial toxin as described above, said at least one aminoacid modification comprises substitution of an acidic amino acid residueor an uncharged amino acid residue with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is anengineered clostridial toxin as described above, said at least one aminoacid modification increases the pI of the engineered clostridial toxinto a value that is at least 0.4 pI units higher than the pI of anotherwise identical clostridial toxin lacking said at least one aminoacid modification. In one embodiment, said at least one amino acidmodification increases the pI of the engineered clostridial toxin to avalue that is at least 0.5 pI units higher than the pI of an otherwiseidentical clostridial toxin lacking said at least one amino acidmodification. In one embodiment, said at least one amino acidmodification increases the pI of the engineered clostridial toxin to avalue that is at least 0.6 pI units higher than the pI of an otherwiseidentical clostridial toxin lacking said at least one amino acidmodification. In one embodiment, said at least one amino acidmodification increases the pI of the engineered clostridial toxin to avalue that is at least 0.8 pI units higher than the pI of an otherwiseidentical clostridial toxin lacking said at least one amino acidmodification. In one embodiment, said at least one amino acidmodification increases the pI of the engineered clostridial toxin to avalue that is at least 1 pI unit higher than the pI of an otherwiseidentical clostridial toxin lacking said at least one amino acidmodification.

In certain embodiments, the engineered clostridial toxin comprises atleast 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85 or 90 amino acid modifications.

In certain embodiments, said at least one amino acid modificationincreases the pI of the engineered clostridial toxin to a value that isat least 2, 3, 4 or 5 pI units higher than the pI of an otherwiseidentical clostridial toxin lacking said at least one amino acidmodification.

In certain embodiments, the engineered clostridial toxin comprises atleast 3 amino acid modifications, and said at least 3 amino acidmodifications increase the pI of the engineered clostridial toxin to avalue that is at least 0.2 pI units higher than the pI of an otherwiseidentical clostridial toxin lacking said at least 3 amino acidmodifications.

In certain embodiments, the engineered clostridial toxin comprises atleast 5 amino acid modifications, and said at least 5 amino acidmodifications increase the pI of the engineered clostridial toxin to avalue that is at least 0.5 pI units higher than the pI of an otherwiseidentical clostridial toxin lacking said at least 5 amino acidmodifications.

The present inventors have found that by increasing the pI of aclostridial toxin, for example, by at least 0.2 pI units, or 0.5 pIunits, or one pI unit (through the introduction into the clostridialtoxin protein of at least one amino acid modification), the resultantengineered clostridial toxin advantageously demonstrates properties ofincreased tissue retention and reduced diffusion away from sites ofadministration, while retaining abilities of target cell binding,translocation, and cleavage of target SNARE protein(s). Thus, the spreadof clostridial toxin from the site of administration is significantlyreduced, as compared to an otherwise identical clostridial toxin lackingsaid at least one amino acid modification.

The engineered clostridial toxins of the invention are suitable for usein any of the therapies described above, and advantageously maydemonstrate a reduction in, or absence of, side effects compared to theuse of known clostridial toxin therapeutics.

The increased tissue retention properties of the engineered clostridialtoxins of the invention also provide increased potency and/or durationof action, and can allow for reduced dosages to be used compared toknown clostridial toxin therapeutics (or increased dosages without anyadditional adverse effects), thus providing further advantages.

Thus, in one embodiment, an engineered clostridial toxin of theinvention has increased potency, increased tissue retention, and/orincreased duration of action, as compared to the correspondingunmodified clostridial toxin.

As discussed below in more detail, the increase in pI provided by the atleast one amino acid modification means that an engineered clostridialtoxin of the invention has, at a given pH, a net charge that is morepositive than the net charge on an otherwise identical clostridial toxinlacking said at least one amino acid modification. Without wishing to bebound by any one theory, the present inventors believe that thisincreased positive charge allows the engineered clostridial toxins ofthe present invention to display longer tissue retention times at thesite of administration due to favourable electrostatic interactionsbetween the engineered clostridial toxin and anionic extracellularcomponents (such as cell membranes and heparin sulphate proteoglycans)at the site of administration. These improved electrostatic interactionsserve to reduce the diffusion of the engineered clostridial toxin awayfrom the site of administration, thus improving tissue retention.

By way of example, the improved tissue retention properties of anengineered clostridial toxin of the invention may allow for (i) higherdoses into individual muscles, such as the sternocleidomastoid, withoutspreading into nearby muscles in the neck to cause difficult swallowing,and (ii) higher total doses (to all muscles) in a single treatment,without spreading into the circulation and causing systemic effects suchas difficult breathing. Advantages to patients may include moreeffective treatment of large muscles such as the sternocleidomastoidmuscle, increased opportunity to inject several different muscles duringeach treatment, and possible longer duration of effective treatment(longer before re-treatment is necessary) because of higher dosing.

In one embodiment, an engineered clostridial toxin of the invention has,in use, a positive net charge (for example, when the engineeredclostridial toxin, in use, is located at a desired administration sitein a tissue).

The isoelectric point (pI) is a specific property of a given protein. Asis well known in the art, proteins are made from a specific sequence ofamino acids (also referred to when in a protein as amino acid residues).Each amino acid of the standard set of twenty has a different side chain(or R group), meaning that each amino acid residue in a protein displaysdifferent chemical properties such as charge and hydrophobicity. Theseproperties may be influenced by the surrounding chemical environment,such as the temperature and pH. The overall chemical characteristics ofa protein will depend on the sum of these various factors.

Certain amino acid residues (detailed below) possess ionisable sidechains that may display an electric charge depending on the surroundingpH. Whether such a side chain is charged or not at a given pH depends onthe pKa of the relevant ionisable moiety, wherein pKa is the negativelogarithm of the acid dissociation constant (Ka) for a specified protonfrom a conjugate base.

For example, acidic residues such as aspartic acid and glutamic acidhave side chain carboxylic acid groups with pKa values of approximately4.1 (precise pKa values may depend on temperature, ionic strength andthe microenvironment of the ionisable group). Thus, these side chainsexhibit a negative charge at a pH of 7.4 (often referred to as“physiological pH”). At low pH values, these side chains will becomeprotonated and lose their charge.

Conversely, basic residues such as lysine and arginine havenitrogen-containing side chain groups with pKa values of approximately10-12. These side chains therefore exhibit a positive charge at a pH of7.4. These side chains will become de-protonated and lose their chargeat high pH values.

The overall (net) charge of a protein molecule therefore depends on thenumber of acidic and basic residues present in the protein (and theirdegree of surface exposure) and on the surrounding pH. Changing thesurrounding pH changes the overall charge on the protein. Accordingly,for every protein there is a given pH at which the number of positiveand negative charges is equal and the protein displays no overall netcharge. This point is known as the isoelectric point (pI). Theisoelectric point is a standard concept in protein biochemistry withwhich the skilled person would be familiar.

The isoelectric point (pI) is therefore defined as the pH value at whicha protein displays a net charge of zero. An increase in pI means that ahigher pH value is required for the protein to display a net charge ofzero. Thus, an increase in pI represents an increase in the net positivecharge of a protein at a given pH.

Conversely, a decrease in pI means that a lower pH value is required forthe protein to display a net charge of zero. Thus, a decrease in pIrepresents a decrease in the net positive charge of a protein at a givenpH.

Methods of determining the pI of a protein are known in the art andwould be familiar to a skilled person. By way of example, the pI of aprotein can be calculated from the average pKa values of each amino acidpresent in the protein (“calculated pI”). Such calculations can beperformed using computer programs known in the art; preferred examplecomputer programs for calculating pI values include Protein Calculatorfrom the Scripps Research Institute and Compute pI/MW Tool from ExPASy.Comparisons of pI values between different molecules should be madeusing the same calculation technique/program.

Where appropriate, the calculated pI of a protein can be confirmedexperimentally using the technique of isoelectric focusing (“observedpI”). This technique uses electrophoresis to separate proteins accordingto their pI. Isoelectric focusing is typically performed using a gelthat has an immobilised pH gradient. When an electric field is applied,the protein migrates through the pH gradient until it reaches the pH atwhich it has zero net charge, this point being the pI of the protein.Results provided by isoelectric focusing are typically relativelylow-resolution in nature, and thus the present inventors believe thatresults provided by calculated pI (as described above) are moreappropriate to use.

Throughout the present specification, “pI” means “calculated pI” unlessotherwise stated.

The pI of a protein may be increased or decreased by altering the numberof basic and/or acidic groups displayed on its surface. This can beachieved by modifying one or more amino acids of the protein. Forexample, an increase in pI may be provided by reducing the number ofacidic residues, or by increasing the number of basic residues. Suchamino acid modifications are discussed in more detail below.

Native (unmodified) clostridial toxins have a pI of approximately 5-6.Thus, at a pH of 7.4, native botulinum toxins possess a negative netcharge. By way of example, the pI of BoNT/A is 6.4, and a BoNT/Amolecule has a net charge at pH 7.4 of −8. These pI values arecalculated as described above.

TABLE 1 CLOSTRIDIAL TOXIN pI BoNT/A 6.4 BoNT/B 5.3 BoNT/C₁ 5.5 BoNT/D5.5 BoNT/E 6.0 BoNT/F 5.6 BoNT/G 5.2 TeNT 5.8

As described above, in one embodiment, an engineered clostridial toxinof the present invention comprises at least one amino acid modification,wherein said at least one amino acid modification increases theisoelectric point (pI) of the engineered clostridial toxin to a valuethat is at least 0.2 pI units higher than the pI of an otherwiseidentical clostridial toxin lacking said at least one amino acidmodification.

Thus, in the context of the present invention, an increase in pI of 0.2units in the context of an engineered BoNT/A clostridial toxin would bean increase in pI from 6.4 to 6.6.

As described above, in one embodiment, an engineered clostridial toxinof the present invention comprises at least one amino acid modification,wherein said at least one amino acid modification increases theisoelectric point (pI) of the engineered clostridial toxin to a valuethat is at least one pI unit higher than the pI of an otherwiseidentical clostridial toxin lacking said at least one amino acidmodification.

Thus, in the context of the present invention, an increase in pI of 1unit in the context of an engineered BoNT/A clostridial toxin would bean increase in pI from 6.4 to 7.4.

In one embodiment, the engineered clostridial toxin has a pI of at least5.5.

In one embodiment, the engineered clostridial toxin has a pI of at least6 (for example, at least 6, at least 7, at least 8, or at least 9).

In one embodiment, the engineered clostridial toxin has a pI of at least6.5.

In one embodiment, the engineered clostridial toxin has a pI of at least7.

In one embodiment, the engineered clostridial toxin has a pI of between6.5 and 9.5 (for example a pI of between 6.5 and 7.5).

As discussed above, the engineered clostridial toxins of the presentinvention have increased tissue retention properties that also provideincreased potency and/or duration of action, and can allow for reduceddosages to be used compared to known clostridial toxin therapeutics (orincreased dosages without any additional effects). One way in whichthese advantageous properties (which represent an increase in thetherapeutic index) may be defined is in terms of the Safety Ratio of theengineered clostridial toxin. In this regard, undesired effects of aclostridial toxin (caused by diffusion of the toxin away from the siteof administration) can be assessed experimentally by measuringpercentage bodyweight loss in a relevant animal model (e.g. a mouse,where loss of bodyweight is detected within seven days ofadministration). Conversely, desired on-target effects of a clostridialtoxin can be assessed experimentally by Digital Abduction Score (DAS)assay, a measurement of muscle paralysis. The DAS assay may be performedby injection of 20 μl of clostridial toxin, formulated in GelatinPhosphate Buffer, into the mouse gastrocnemius/soleus complex, followedby assessment of Digital Abduction Score using the method of Aoki (AokiKR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are suspendedbriefly by the tail in order to elicit a characteristic startle responsein which the mouse extends its hind limbs and abducts its hind digits.Following clostridial toxin injection, the varying degrees of digitabduction are scored on a five-point scale (0=normal to 4=maximalreduction in digit abduction and leg extension).

The Safety Ratio of a clostridial toxin may then be expressed as theratio between the amount of toxin required for a 10% drop in abodyweight (measured at peak effect within the first seven days afterdosing in a mouse) and the amount of toxin required for a DAS score of2. High Safety Ratio scores are therefore desired, and indicate a toxinthat is able to effectively paralyse a target muscle with littleundesired off-target effects. An engineered toxin of the presentinvention has a Safety Ratio that is higher than the Safety Ratio of anequivalent unmodified (native) botulinum toxin.

Thus, in one embodiment, an engineered clostridial toxin of the presentinvention has a Safety Ratio of at least 8 (for example, at least 8, 9,10, 15, 20, 25, 30, 35, 40, 45 or 50), wherein Safety Ratio iscalculated as: dose of toxin required for −10% bodyweight change(pg/mouse) divided by DAS ED₅₀ (pg/mouse) [ED₅₀=dose required to producea DAS score of 2].

In one embodiment, an engineered clostridial toxin of the presentinvention has a Safety Ratio of at least 10. In one embodiment, anengineered clostridial toxin of the present invention has a Safety Ratioof at least 15.

An engineered clostridial toxin of the present invention comprises atleast one amino acid modification. Said at least one amino acidmodification increases the pI of the clostridial toxin, as discussedabove. In the context of the present invention, an amino acidmodification is a modification of the amino acid sequence of aclostridial toxin. Such a modification may be effected by replacing oneamino acid in the sequence with another (i.e. a substitution), byinserting a new amino acid into the sequence, or by deleting an aminoacid of the sequence. Amino acids incorporated into an amino acidsequence in a protein are also referred to as amino acid residues.

The 20 standard amino acids found in proteins are as follows:

TABLE 2 AMINO ACID SIDE CHAIN Aspartic acid Asp D Charged (acidic)Glutamic acid Glu E Charged (acidic) Arginine Arg R Charged (basic)Lysine Lys K Charged (basic) Histidine His H Uncharged (polar)Asparagine Asn N Uncharged (polar) Glutamine Gln Q Uncharged (polar)Serine Ser S Uncharged (polar) Threonine Thr T Uncharged (polar)Tyrosine Tyr Y Uncharged (polar) Methionine Met M Uncharged (polar)Tryptophan Trp W Uncharged (polar) Cysteine Cys C Uncharged (polar)Alanine Ala A Uncharged (hydrophobic) Glycine Gly G Uncharged(hydrophobic) Valine Val V Uncharged (hydrophobic) Leucine Leu LUncharged (hydrophobic) Isoleucine Ile I Uncharged (hydrophobic) ProlinePro P Uncharged (hydrophobic) Phenylalanine Phe F Uncharged(hydrophobic)

The following amino acids are considered charged amino acids: asparticacid (negative), glutamic acid (negative), arginine (positive), andlysine (positive).

At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamicacid (pKa 4.1) have a negative charge, while the side chains of arginine(pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acidand glutamic acid are referred to as acidic amino acid residues.Arginine and lysine are referred to as basic amino acid residues.

The following amino acids are considered uncharged, polar (meaning theycan participate in hydrogen bonding) amino acids: asparagine, glutamine,histidine, serine, threonine, tyrosine, cysteine, methionine,tryptophan.

The following amino acids are considered uncharged, hydrophobic aminoacids: alanine, valine, leucine, isoleucine, phenylalanine, proline, andglycine.

An increase in the pI of a clostridial toxin can be effected byintroducing into the clostridial toxin one or more amino acidmodifications that increases the ratio of positive to negative chargesin the clostridial toxin.

In one embodiment, the at least one amino acid modification is selectedfrom: an amino acid substitution, an amino acid insertion, and an aminoacid deletion.

In an amino acid substitution, an amino acid residue that forms part ofthe clostridial toxin amino acid sequence is replaced with a differentamino acid residue. The replacement amino acid residue may be one of the20 standard amino acids, as described above.

Alternatively, the replacement amino acid in an amino acid substitutionmay be a non-standard amino acid (an amino acid that is not part of thestandard set of 20 described above). By way of example, the replacementamino acid may be a basic non-standard amino acid, e.g. L-Ornithine,L-2-amino-3-guanidinopropionic acid, or D-isomers of Lysine, Arginineand Ornithine). Methods for introducing non-standard amino acids intoproteins are known in the art, and include recombinant protein synthesisusing E. coli auxotrophic expression hosts.

In an amino acid insertion, an additional amino acid residue (one thatis not normally present) is incorporated into the clostridial toxinamino acid sequence, thus increasing the total number of amino acidresidues in said sequence. In an amino acid deletion, an amino acidresidue is removed from the clostridial toxin amino acid sequence, thusreducing the total number of amino acid residues in said sequence.

Methods for modifying proteins by substitution, insertion or deletion ofamino acid residues are known in the art. By way of example, amino acidmodifications may be introduced by modification of a DNA sequenceencoding a clostridial toxin. This can be achieved using standardmolecular cloning techniques, for example by site-directed mutagenesiswhere short strands of DNA (oligonucleotides) coding for the desiredamino acid(s) are used to replace the original coding sequence using apolymerase enzyme, or by inserting/deleting parts of the gene withvarious enzymes (e.g., ligases and restriction endonucleases).Alternatively a modified gene sequence can be chemically synthesised.

In one embodiment, the at least one amino acid modification is selectedfrom: substitution of an acidic amino acid residue with a basic aminoacid residue; substitution of an acidic amino acid residue with anuncharged amino acid residue; substitution of an uncharged amino acidresidue with a basic amino acid residue; insertion of a basic amino acidresidue; and deletion of an acidic amino acid residue.

In a preferred embodiment, the at least one amino acid modification is asubstitution, which advantageously maintains the same number of aminoacid residues in the clostridial toxin. In one embodiment, thesubstitution is selected from: substitution of an acidic amino acidresidue with a basic amino acid residue, substitution of an acidic aminoacid residue with an uncharged amino acid residue, and substitution ofan uncharged amino acid residue with a basic amino acid residue. In oneembodiment, the basic amino acid residue is a lysine residue or anarginine residue. In one embodiment, the basic amino acid residue is alysine residue. In one embodiment, the basic amino acid residue is anarginine residue. In one embodiment, wherein the substitution is asubstitution of an acidic amino acid residue with an uncharged aminoacid residue, the acidic amino acid residue is replaced with itscorresponding uncharged amide amino acid residue (i.e. aspartic acid isreplaced with asparagine, and glutamic acid is replaced with glutamine).

In another preferred embodiment, the at least one amino acidmodification is a substitution of an acidic amino acid residue with abasic amino acid residue, or a substitution of an uncharged amino acidresidue with a basic amino acid residue. In one embodiment, the basicamino acid residue is a lysine residue. In one embodiment, the basicamino acid residue is an arginine residue.

An engineered clostridial toxin of the invention may comprise more thanone amino acid modification. Thus, in one embodiment, the engineeredclostridial toxin (as described above) comprises between 1 and 90 aminoacid modifications (for example, between 1 and 80, between 1 and 70,between 1 and 60, between 1 and 50, between 1 and 40, between 1 and 30,between 1 and 20, between 1 and 10, between 3 and 50, between 3 and 40,between 3 and 30, between 4 and 40, between 4 and 30, between 5 and 40,between 5 and 30, or between 10 and 25 amino acid modifications). In oneembodiment, the engineered clostridial toxin (as described above)comprises between 1 and 20 amino acid modifications. In one embodiment,the engineered clostridial toxin (as described above) comprises between1 and 10 amino acid modifications. In one embodiment, the engineeredclostridial toxin (as described above) comprises between 2 and 20 aminoacid modifications. In one embodiment, the engineered clostridial toxin(as described above) comprises between 2 and 15 amino acidmodifications. In one embodiment, the engineered clostridial toxin (asdescribed above) comprises between 2 and 10 amino acid modifications. Inone embodiment, the engineered clostridial toxin (as described above)comprises between 3 and 20 amino acid modifications. In one embodiment,the engineered clostridial toxin (as described above) comprises between3 and 15 amino acid modifications. In one embodiment, the engineeredclostridial toxin (as described above) comprises between 3 and 10 aminoacid modifications. In one embodiment, the engineered clostridial toxin(as described above) comprises between 4 and 40 amino acidmodifications. In one embodiment, the engineered clostridial toxincomprises at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10 amino acidmodifications. In one embodiment, the engineered clostridial toxincomprises at least 3 amino acid modifications (for example, at least 3amino acid substitutions). In one embodiment, the engineered clostridialtoxin comprises at least 4 amino acid modifications (for example, atleast 4 amino acid substitutions). Each of said amino acid modificationsis an amino acid modification as described above. Thus, each of saidamino acid modifications contributes to the increase in pI of theengineered clostridial toxin (as compared to the pI of an otherwiseidentical clostridial toxin lacking said amino acid modifications).

Any clostridial toxin amino acid (i.e. amino acid residue) that is notlocated in the clostridial toxin binding domain (H_(C) domain) can bemodified as described above, as long as the outcome of said modificationis an increase in the clostridial toxin pI (as described above).However, the present inventors have identified subsets of clostridialtoxin amino acids that are particularly suitable targets formodification.

Preferred target amino acids may possess certain qualities. By way ofexample, a preferred target amino acid may be: (i) a surface exposedamino acid; (ii) located outside of a clostridial toxin proteinsecondary structure; (iii) located in a clostridial toxin protein regionthat is non-essential for protein function; (iv) an amino acid whoseidentity is not conserved between clostridial toxin types, subtypes, orserotypes; (iv) an amino acid whose modification does not create apredicted ubiquitination site; or (v) any combination of the foregoing.

As described above, the engineered clostridial toxins of the inventionfeature one or more amino acid modifications located in either theclostridial toxin H_(N) translocation domain or the clostridial toxinlight chain.

Examples of clostridial toxin light chain reference sequences include:

Botulinum type A neurotoxin: amino acid residues 1-448Botulinum type B neurotoxin: amino acid residues 1-440Botulinum type C₁ neurotoxin: amino acid residues 1-441Botulinum type D neurotoxin: amino acid residues 1-445Botulinum type E neurotoxin: amino acid residues 1-422Botulinum type F neurotoxin: amino acid residues 1-439Botulinum type G neurotoxin: amino acid residues 1-441Tetanus neurotoxin: amino acid residues 1-457

Examples of clostridial toxin H_(N) domain reference sequences include:

Botulinum type A neurotoxin: amino acid residues 449-871Botulinum type B neurotoxin: amino acid residues 443-862Botulinum type C₁ neurotoxin: amino acid residues 450-866Botulinum type D neurotoxin: amino acid residues 449-871Botulinum type E neurotoxin: amino acid residues 455-845Botulinum type F neurotoxin: amino acid residues 450-864Botulinum type G neurotoxin: amino acid residues 449-871Tetanus neurotoxin: amino acid residues 456-879

Examples of clostridial toxin H_(C) domain reference sequences include:

Botulinum type A neurotoxin: amino acid residues 872-1278Botulinum type B neurotoxin: amino acid residues 863-1291Botulinum type C₁ neurotoxin: amino acid residues 867-1291Botulinum type D neurotoxin: amino acid residues 872-1276Botulinum type E neurotoxin: amino acid residues 846-1252Botulinum type F neurotoxin: amino acid residues 865-1278Botulinum type G neurotoxin: amino acid residues 872-1297Tetanus neurotoxin: amino acid residues 880-1315

The above-identified reference sequences should be considered a guide,as slight variations may occur according to sub-serotypes. By way ofexample, US 2007/0166332 (hereby incorporated by reference in itsentirety) cites slightly different clostridial sequences:

Light chain:

Botulinum type A neurotoxin: amino acid residues M1-K448Botulinum type B neurotoxin: amino acid residues M1-K441Botulinum type C₁ neurotoxin: amino acid residues M1-K449Botulinum type D neurotoxin: amino acid residues M1-R445Botulinum type E neurotoxin: amino acid residues M1-R422Botulinum type F neurotoxin: amino acid residues M1-K439Botulinum type G neurotoxin: amino acid residues M1-K446 Tetanusneurotoxin: amino acid residues M1-A457

H_(N) domain:

Botulinum type A neurotoxin: amino acid residues A449-K871Botulinum type B neurotoxin: amino acid residues A442-S858Botulinum type C₁ neurotoxin: amino acid residues T450-N866Botulinum type D neurotoxin: amino acid residues D446-N862Botulinum type E neurotoxin: amino acid residues K423-K845Botulinum type F neurotoxin: amino acid residues A440-K864Botulinum type G neurotoxin: amino acid residues S447-S863 Tetanusneurotoxin: amino acid residues S458-V879

He domain:

Botulinum type A neurotoxin: amino acid residues N872-L1296Botulinum type B neurotoxin: amino acid residues E859-E1291Botulinum type C₁ neurotoxin: amino acid residues N867-E1291Botulinum type D neurotoxin: amino acid residues S863-E1276Botulinum type E neurotoxin: amino acid residues R846-K1252Botulinum type F neurotoxin: amino acid residues K865-E1274Botulinum type G neurotoxin: amino acid residues N864-E1297 Tetanusneurotoxin: amino acid residues I880-D1315

In one embodiment, wherein said at least one amino acid modification islocated in the clostridial toxin translocation domain (H_(N) domain),said at least one amino acid modification is not located in theclostridial toxin belt region. The clostridial toxin belt region (asdetermined by visual inspection of structures and models) is defined asfollows:

Botulinum type A neurotoxin: amino acid residues 492-545Botulinum type B neurotoxin: amino acid residues 472-532Botulinum type C₁ neurotoxin: amino acid residues 494-543Botulinum type D neurotoxin: amino acid residues 489-539Botulinum type E neurotoxin: amino acid residues 466-515Botulinum type F neurotoxin: amino acid residues 485-536Botulinum type G neurotoxin: amino acid residues 489-538 Tetanusneurotoxin: amino acid residues 506-556

In one embodiment, the at least one amino acid modification (asdescribed above) is a modification of a surface exposed amino acidresidue. Surface exposed amino acid residues are those present on theexterior of a folded protein and so accessible to the surroundingsolvent, in contrast to those amino acid residues that are located inthe interior of a folded protein. The degree of surface exposure of anamino acid residue and thus its exposure to the surrounding solventdepends on its position within the folded protein, and also on theconformation adopted by the protein. Modification of an amino acidresidue with a high degree of surface exposure may therefore have agreater effect on the protein's isoelectric point than modification ofan amino acid residue with a low degree of surface exposure. Methods fordetermining the degree of surface exposure of an amino acid residue areknown in the art. By way of example, the computer program AreaIMol (partof the CCP4 suite of computer programs) can be used to calculate thedegree of surface exposure of amino acid residues in a given protein.Surface exposed amino acid residues may also be identified by visualinspection of a protein crystal structure (such as provided by X-raycrystallography). In one embodiment, a surface exposed amino acidresidue has a sum AreaIMol value of at least 40.

In one embodiment, the at least one amino acid modification comprisesmodification of an amino acid residue selected from: an aspartic acidresidue, a glutamic acid residue, a histidine residue, an asparagineresidue, a glutamine residue, a serine residue, a threonine residue, analanine residue, a glycine residue, a valine residue, a leucine residue,and an isoleucine residue. The present inventors have identified thatamino acid residues from this group represent particularly suitabletargets for modification according to the present invention.

In one embodiment, wherein the amino acid modification comprisesmodification of an amino acid residue selected from an aspartic acidresidue, a glutamic acid residue, a histidine residue, an asparagineresidue, a glutamine residue, a serine residue, a threonine residue, analanine residue, a glycine residue, a valine residue, a leucine residue,and an isoleucine residue (as described above), the amino acid residueis substituted with a lysine residue or an arginine residue. In oneembodiment, the amino acid residue is substituted with an arginineresidue. Thus, in one embodiment, a negatively charged residue, a polarresidue, or an uncharged residue is substituted with a positivelycharged residue, thus increasing the ratio of positive to negativecharges and increasing the pI of the clostridial toxin.

In one embodiment, the at least one amino acid modification (asdescribed above) comprises modification of an asparagine amino acidresidue or a glutamine amino acid residue (both uncharged, polarresidues). In one embodiment, the asparagine or glutamine amino acidresidue is substituted with a lysine residue or an arginine residue(both positively charged residues). In one embodiment, the asparagine orglutamine amino acid residue is substituted with a lysine residue. Inone embodiment, the asparagine or glutamine amino acid residue issubstituted with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/A. Areference BoNT/A sequence has the UniProtKB Accession Number P10845. Inone embodiment wherein the engineered clostridial toxin is a BoNT/A, theengineered clostridial toxin is a BoNT/A1

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/A clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A,said engineered BoNT/A comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 orall 55) amino acid selected from: D474, N476, D484, N486, I487, E488,A489, A490, E491, D546, E558, E560, H561, I566, L568, N570, S571, L577,N578, A597, E599, A601, E620, V621, T623, D625, T631, N645, L647, D650,D651, I668, E670, A672, V675, S683, I685, A686, N687, N752, Q753, T755,E756, E757, E758, N760, N761, I762, N763, D825, I831, G832, T847, D848,and D858; and said amino acid modification(s) increase(s) theisoelectric point (pI) of the engineered BoNT/A to a value that is atleast 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9or 1) pI units higher than the pI of an otherwise identical BoNT/Alacking said amino acid modification(s). In one embodiment saidmodification comprises substitution of the amino acid with a lysineresidue or an arginine residue. In one embodiment, said modificationcomprises substitution of the amino acid with a lysine residue. In oneembodiment, said modification comprises substitution of the amino acidwith an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A,said engineered BoNT/A comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15 or all 17) amino acid selectedfrom: N476, S564, N578, E599, L647, D650, D651, V675, I685, N687, T755,E757, N761, N763, I831, T847, and 1849; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/A lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A,said engineered BoNT/A comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5 or all 6) amino acid selected from:S564, L647, D650, D651, T847, and 1849; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/A lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A,said engineered BoNT/A comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5 or all 6) amino acid selected from:N476, N763, N687, E599, I831, and N761; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/A lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A,said engineered BoNT/A comprises a modification of at least one (forexample, at least 1, 2, 3, 4, or all 5) amino acid selected from: N578,V675, I685, T755, and E757; and said amino acid modification(s)increase(s) the isoelectric point (pI) of the engineered BoNT/A to avalue that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of an otherwiseidentical BoNT/A lacking said amino acid modification(s). In oneembodiment said modification comprises substitution of the amino acidwith a lysine residue or an arginine residue. In one embodiment, saidmodification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/B. Areference BoNT/B sequence has the UniProtKB Accession Number P10844.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/B clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/B,said engineered BoNT/B comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or all 40)amino acid selected from: V443, G444, D453, S468, D533, E534, N535,T545, L548, D549, I550, D552, S557, L564, S566, N582, V584, N609, L619,N632, E633, G637, A646, I655, E657, V662, E669, S670, I672, D673, N739,I740, N748, N750, I818, G819, T834, I842, N845, and 5858; and said aminoacid modification(s) increase(s) the isoelectric point (pI) of theengineered BoNT/B to a value that is at least 0.2 (for example, at least0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pIof an otherwise identical BoNT/B lacking said amino acidmodification(s). In one embodiment said modification comprisessubstitution of the amino acid with a lysine residue or an arginineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment, saidmodification comprises substitution of the amino acid with an arginineresidue.

In one embodiment, the engineered clostridial toxin is a BoNT/C₁. Areference BoNT/C₁ sequence has the UniProtKB Accession Number P18640.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/C₁ clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is aBoNT/C₁, said engineered BoNT/C₁ comprises a modification of at leastone (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45or all 50) amino acid selected from: L451, D452, C453, E455, V472, T474,D475, L478, N483, E484, E485, E487, I489, L555, S556, D557, N558, E560,D561, E569, N574, S575, T584, G592, Q594, G596, D617, N640, S641, V642,G645, N646, E661, E665, T667, A670, S678, V680, Q681, E682, S750, G751,S759, Q760, V826, G827, N842, T843, N847, and N853; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/C₁ to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/C₁ lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/D. Areference BoNT/D sequence has the UniProtKB Accession Number P19321.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/D clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/D,said engineered BoNT/D comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, or all 62) amino acid selected from: Q469, E470, E473, N474, D479,E480, N482, V483, Q484, N485, S487, D488, S552, N553, N554, V555, E556,N557, I558, L560, T562, S563, V564, G569, S571, N572, G588, Q590, T614,D616, S619, S622, N636, S637, L639, G641, N642, E657, E661, T663, A666,V669, S674, I676, Q677, E678, S746, G747, D749, E751, N752, I753, Q756,N818, V822, G823, E837, N838, T839, N843, N849, and N850; and said aminoacid modification(s) increase(s) the isoelectric point (pI) of theengineered BoNT/D to a value that is at least 0.2 (for example, at least0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pIof an otherwise identical BoNT/B lacking said amino acidmodification(s). In one embodiment said modification comprisessubstitution of the amino acid with a lysine residue or an arginineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment, saidmodification comprises substitution of the amino acid with an arginineresidue.

In one embodiment, the engineered clostridial toxin is a BoNT/E. Areference BoNT/E sequence has the UniProtKB Accession Number Q00496. Inone embodiment wherein the engineered clostridial toxin is a BoNT/E, theengineered clostridial toxin is a BoNT/E3.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/E clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,said engineered BoNT/E comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, orall 52) amino acid selected from: D474, N476, E479, E480, D484, N486,I487, E488, A489, A490, E491, E492, L496, D497, Q500, Q501, L504, N507,D509, N510, N514, S516, E518, Q527, L530, N533, I534, E535, N539, Y548,I566, L568, D589, A597, E599, A601, L604, Y612, E620, N645, L647, Y648,D651, E737, E741, Y803, Y824, D825, G828, I831, G832, and D835; and saidamino acid modification(s) increase(s) the isoelectric point (pI) of theengineered BoNT/E to a value that is at least 0.2 (for example, at least0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pIof an otherwise identical BoNT/E lacking said amino acidmodification(s). In one embodiment said modification comprisessubstitution of the amino acid with a lysine residue or an arginineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment, saidmodification comprises substitution of the amino acid with an arginineresidue.

In one embodiment, the engineered clostridial toxin is a BoNT/F. Areference BoNT/F sequence has the UniProtKB Accession NumberYP_001390123.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/F clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/F,said engineered BoNT/F comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, or all 86) amino acid selected from: N463, E464,N468, T469, D474, D475, T476, T477, N478, N482, N485, N495, I499, Q501,I502, Q505, T506, N508, T509, V511, D513, D521, S522, S526, E527, I528,E529, V534, D535, L536, E549, G550, T552, N553, S558, E566, E567, S568,V586, H587, Q608, D613, A616, D617, S619, N630, N633, N639, E654, V656,E658, L660, T663, L665, V666, S671, I673, G674, S675, S676, E677, N678,T746, N751, L753, E754, E756, N758, I759, N760, N761, S799, S821, I822,N840, S841, E845, L846, S847, S848, T850, N851, D852, I854, L855, and1856; and said amino acid modification(s) increase(s) the isoelectricpoint (pI) of the engineered BoNT/F to a value that is at least 0.2 (forexample, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI unitshigher than the pI of an otherwise identical BoNT/F lacking said aminoacid modification(s). In one embodiment said modification comprisessubstitution of the amino acid with a lysine residue or an arginineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment, saidmodification comprises substitution of the amino acid with an arginineresidue.

In one embodiment, the engineered clostridial toxin is a BoNT/G. Areference BoNT/G sequence has the UniProtKB Accession Number Q60393.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/G clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/G,said engineered BoNT/G comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or all 36)amino acid selected from: N480, Q482, N483, N484, T485, E487, D540,N562, N570, N571, N572, T588, V589, T615, D621, N637, E638, E642, N643,I660, E662, I667, E674, S675, V677, G678, N679, S747, N755, D757, L823,D839, I841, D844, S846, and L847; and said amino acid modification(s)increase(s) the isoelectric point (pI) of the engineered BoNT/G to avalue that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of an otherwiseidentical BoNT/G lacking said amino acid modification(s). In oneembodiment said modification comprises substitution of the amino acidwith a lysine residue or an arginine residue. In one embodiment, saidmodification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a TeNT. Areference TeNT sequence has the UniProtKB Accession Number P04958.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a TeNT clostridialtoxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a TeNT,said engineered TeNT comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or all49) amino acid selected from: A457, S458, L459, D461, L462, E486, E487,Q490, D491, N497, N504, D557, T571, T572, L573, Q574, N580, S581, N588,S589, T590, S598, Q605, G606, Q608, T631, I633, S640, Q655, E658, G659,N660, E675, I677, E679, T681, V684, A691, E692, S694, T695, Q696, A772,D773, E774, S862, N866, L867 and D868; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredTeNT to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical TeNT lacking said amino acid modification(s). In oneembodiment said modification comprises substitution of the amino acidwith a lysine residue or an arginine residue. In one embodiment, saidmodification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/A clostridialtoxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A,said engineered BoNT/A comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or all 28) amino acidselected from: N5, Q7, N9, D12, N15, Q31, D58, N60, D74, N82, T122,D124, E126, Q139, D141, E281, L284, S295, Q311, D326, D334, N377,TYR387, N394, N396, N410, M411, and N418; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/A lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/B clostridialtoxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/B,said engineered BoNT/B comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40 or all 41amino acid selected from: N6, N7, N9, N11, D12, N16, N17, N18, D41, E48,E57, N60, D75, D77, N80, E127, N130, N144, E147, E149, E185, N216, D245,E253, N316, D333, E335, D341, N385, D388, N389, E390, E395, E396, D402,D404, E406, E408, Q419, E423, and E427; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/B to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/B lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/C₁ clostridialtoxin light chain.

in one embodiment, wherein the engineered clostridial toxin is aBoNT/C₁, said engineered BoNT/C₁ comprises a modification of at leastone (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or all 41)amino acid selected from: N6, N7, N9, D12, D15, N18, N31, E32, N55, N59,N75, N120, N121, N125, D128, Q142, N145, N177, N178, Q183, E184, D208,E211, Q247, N255, N311, E335, E339, N343, N368, N386, D389, D390, N391,Q396, N405, N407, N425, E427, D442, and N448; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/C₁ to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/C₁ lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/D clostridialtoxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/D,said engineered BoNT/D comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or all 34) amino acidselected from: D7, N9, D12, N15, D16, N17, D53, D73, D119, E124, E139,E142, N143, Q177, Q178, N180, E184, E255, N308, D335, N336, N339, N343,N368, N386, D389, D390, N391, D397, N403, N407, E409, E416, and N443;and said amino acid modification(s) increase(s) the isoelectric point(pI) of the engineered BoNT/D to a value that is at least 0.2 (forexample, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI unitshigher than the pI of an otherwise identical BoNT/D lacking said aminoacid modification(s). In one embodiment said modification comprisessubstitution of the amino acid with a lysine residue or an arginineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment, saidmodification comprises substitution of the amino acid with an arginineresidue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/E clostridialtoxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,said engineered BoNT/E comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or all 37) aminoacid selected from: N5, N8, N10, D11, N14, D15, Q27, E28, Q53, N72, Q75,D117, N118, D121, N122, Q123, N138, N169, N170, N195, Q237, ILE244,Q290, N293, N297, D312, Q344, N362, N365, D366, N370, E373, N378, N379,N383, N390, and T397; and said amino acid modification(s) increase(s)the isoelectric point (pI) of the engineered BoNT/E to a value that isat least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9 or 1) pI units higher than the pI of an otherwise identical BoNT/Elacking said amino acid modification(s). In one embodiment saidmodification comprises substitution of the amino acid with a lysineresidue or an arginine residue. In one embodiment, said modificationcomprises substitution of the amino acid with a lysine residue. In oneembodiment, said modification comprises substitution of the amino acidwith an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,said engineered BoNT/E comprises a modification of a least one (forexample, at least 1, 2, 3, 4, 5, 10, 15 or 20) amino acid selected from:N5, N8, N10, D11, N14, D15, Q27, E28, N72, Q75, N118, D121, N122, Q123,N138, Q237, Q290, N297, N362, N365, D366, N378, and N379; and said aminoacid modification(s) increase(s) the isoelectric point (pI) of theengineered BoNT/E to a value that is at least 0.2 (for example, at least0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pIof an otherwise identical BoNT/E lacking said amino acidmodification(s). In one embodiment said modification comprisessubstitution of the amino acid with a lysine residue or an arginineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment, saidmodification comprises substitution of the amino acid with an arginineresidue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,said engineered BoNT/E comprises a modification of a least one (forexample, at least 1, 2, 3, 4, 5, 10, 15 or all 19) amino acid selectedfrom: N5, N8, N10, D11, N14, D15, N72, Q75, N118, N122, Q123, N138,Q237, Q290, Q297, N362, D366, N378, and N379; and said amino acidmodification(s) increase(s) the isoelectric point (pI) of the engineeredBoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of anotherwise identical BoNT/E lacking said amino acid modification(s). Inone embodiment said modification comprises substitution of the aminoacid with a lysine residue or an arginine residue. In one embodiment,said modification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,said engineered BoNT/E comprises a modification of a least one (forexample, at least 1, 2, 3, 4, 5, or all 6) amino acid selected from: N8,N10, Q75, Q123, N138, and Q237; and said amino acid modification(s)increase(s) the isoelectric point (pI) of the engineered BoNT/E to avalue that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9 or 1) pI units higher than the pI of an otherwiseidentical BoNT/E lacking said amino acid modification(s). In oneembodiment said modification comprises substitution of the amino acidwith a lysine residue or an arginine residue. In one embodiment, saidmodification comprises substitution of the amino acid with a lysineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E,said engineered BoNT/E comprises a modification of a least one (forexample, at least 1, 2, or all 3) amino acid selected from: Q123, N138,and Q237; and said amino acid modification(s) increase(s) theisoelectric point (pI) of the engineered BoNT/E to a value that is atleast 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9or 1) pI units higher than the pI of an otherwise identical BoNT/Elacking said amino acid modification(s). In one embodiment saidmodification comprises substitution of the amino acid with a lysineresidue or an arginine residue. In one embodiment, said modificationcomprises substitution of the amino acid with a lysine residue. In oneembodiment, said modification comprises substitution of the amino acidwith an arginine residue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/F clostridialtoxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/F,said engineered BoNT/F comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or all 35) amino acidselected from: N6, N9, N11, D12, N15, D16, D17, E28, D55, D60, D74, N76,E105, E121, N126, E127, N144, D185, N211, Q252, N305, E310, D312, N314,N329, D331, N379, D382, D383, D384, E390, N396, N400, D414, and D418;and said amino acid modification(s) increase(s) the isoelectric point(pI) of the engineered BoNT/F to a value that is at least 0.2 (forexample, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pI unitshigher than the pI of an otherwise identical BoNT/F lacking said aminoacid modification(s). In one embodiment said modification comprisessubstitution of the amino acid with a lysine residue or an arginineresidue. In one embodiment, said modification comprises substitution ofthe amino acid with a lysine residue. In one embodiment, saidmodification comprises substitution of the amino acid with an arginineresidue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a BoNT/G clostridialtoxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/G,said engineered BoNT/G comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or all 38) aminoacid selected from: N4, N7, N9, N11, D12, N15, D17, E48, Q55, D57, N60,D75, D127, Q144, E148, D149, Q150, N178, E185, E208, D211, E255, D315,D332, N334, D340, E383, D387, N388, Q393, N394, E395, N403, E407, E418,E422, E426, and N443; and said amino acid modification(s) increase(s)the isoelectric point (pI) of the engineered BoNT/G to a value that isat least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9 or 1) pI units higher than the pI of an otherwise identical BoNT/Glacking said amino acid modification(s). In one embodiment saidmodification comprises substitution of the amino acid with a lysineresidue or an arginine residue. In one embodiment, said modificationcomprises substitution of the amino acid with a lysine residue. In oneembodiment, said modification comprises substitution of the amino acidwith an arginine residue.

The present inventors have identified certain amino acids that representpreferred targets for amino acid modification in a TeNT light chain.

In one embodiment, wherein the engineered clostridial toxin is a TeNT,said engineered TeNT comprises a modification of at least one (forexample, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or all 36) aminoacid selected from: N6, N7, N15, N16, D17, D31, E51, E57, N60, N76,N101, D126, D143, N167, D179, N180, E251, Q257, N313, N316, D318, D335,N337, Q339, N368, N387, D390, D391, N395, D396, E403, D406, E410, N421,D427, and E450; and said amino acid modification(s) increase(s) theisoelectric point (pI) of the engineered TeNT to a value that is atleast 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9or 1) pI units higher than the pI of an otherwise identical TeNT lackingsaid amino acid modification(s). In one embodiment said modificationcomprises substitution of the amino acid with a lysine residue or anarginine residue. In one embodiment, said modification comprisessubstitution of the amino acid with a lysine residue. In one embodiment,said modification comprises substitution of the amino acid with anarginine residue.

The present invention is suitable for application to many differentvarieties of clostridial toxin. Thus, in the context of the presentinvention, the term “clostridial toxin” embraces toxins produced by C.botulinum (botulinum neurotoxin serotypes A, B, C₁, D, E, F and G), C.tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotypeE), and C. baratii (botulinum neurotoxin serotype F), as well asmodified clostridial toxins or derivatives derived from any of theforegoing. The term “clostridial toxin” also embraces botulinumneurotoxin serotype H.

Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of alarge protein complex, consisting of BoNT itself complexed to a numberof accessory proteins. There are at present eight different classes ofbotulinum neurotoxin, namely: botulinum neurotoxin serotypes A, B, C₁,D, E, F, G, and H, all of which share similar structures and modes ofaction. Different BoNT serotypes can be distinguished based oninactivation by specific neutralising anti-sera, with suchclassification by serotype correlating with percentage sequence identityat the amino acid level. BoNT proteins of a given serotype are furtherdivided into different subtypes on the basis of amino acid percentagesequence identity.

BoNTs are absorbed in the gastrointestinal tract, and, after enteringthe general circulation, bind to the presynaptic membrane of cholinergicnerve terminals and prevent the release of their neurotransmitteracetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleavesynaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/C₁,BoNT/A and BoNT/E cleave the synaptosomal-associated protein of 25 kDa(SNAP-25); and BoNT/C₁ cleaves syntaxin.

Tetanus toxin is produced in a single serotype by C. tetani. C.butyricum produces BoNT/E, while C. baratii produces BoNT/F.

The term “clostridial toxin” is also intended to embrace modifiedclostridial toxins and derivatives thereof, including but not limited tothose described below. A modified clostridial toxin or derivative maycontain one or more amino acids that has been modified as compared tothe native (unmodified) form of the clostridial toxin, or may containone or more inserted amino acids that are not present in the native(unmodified) form of the clostridial toxin. By way of example, amodified clostridial toxin may have modified amino acid sequences in oneor more domains relative to the native (unmodified) clostridial toxinsequence. Such modifications may modify functional aspects of the toxin,for example biological activity or persistence. Thus, in one embodiment,the engineered clostridial toxin of the invention is an engineeredmodified clostridial toxin, or an engineered modified clostridial toxinderivative, or an engineered clostridial toxin derivative.

A modified clostridial toxin may have one or more modifications in theamino acid sequence of the heavy chain (such as a modified H_(C)domain), wherein said modified heavy chain binds to target nerve cellswith a higher or lower affinity than the native (unmodified) clostridialtoxin. Such modifications in the H_(C) domain can include modifyingresidues in the ganglioside binding site of the H_(C) domain or in theprotein (SV2 or synaptotagmin) binding site that alter binding to theganglioside receptor and/or the protein receptor of the target nervecell. Examples of such modified clostridial toxins are described in WO2006/027207 and WO 2006/114308, both of which are hereby incorporated byreference in their entirety.

A modified clostridial toxin may have one or more modifications in theamino acid sequence of the light chain, for example modifications in thesubstrate binding or catalytic domain which may alter or modify theSNARE protein specificity of the modified LC. Examples of such modifiedclostridial toxins are described in WO 2010/120766 and US 2011/0318385,both of which are hereby incorporated by reference in their entirety.

A modified clostridial toxin may comprise one or more modifications thatincreases or decreases the biological activity and/or the biologicalpersistence of the modified clostridial toxin. For example, a modifiedclostridial toxin may comprise a leucine- or tyrosine-based motif,wherein said motif increases or decreases the biological activity and/orthe biological persistence of the modified clostridial toxin. Suitableleucine-based motifs include xDxxxLL, xExxxLL, xExxxLL, and xExxxLM(wherein x is any amino acid). Suitable tyrosine-based motifs includeY-x-x-Hy (wherein Hy is a hydrophobic amino acid). Examples of modifiedclostridial toxins comprising leucine- and tyrosine-based motifs aredescribed in WO 2002/08268, which is hereby incorporated by reference inits entirety.

The term “clostridial toxin” is intended to embrace hybrid and chimericclostridial toxins. A hybrid clostridial toxin comprises at least aportion of a light chain from one clostridial toxin or subtype thereof,and at least a portion of a heavy chain from another clostridial toxinor clostridial toxin subtype. In one embodiment the hybrid clostridialtoxin may contain the entire light chain from one clostridial toxinsubtype and the heavy chain from another clostridial toxin subtype. Inanother embodiment, a chimeric clostridial toxin may contain a portion(e.g. the binding domain) of the heavy chain of one clostridial toxinsubtype, with another portion of the heavy chain being from anotherclostridial toxin subtype. Similarly or alternatively, the therapeuticelement may comprise light chain portions from different clostridialtoxins. Such hybrid or chimeric clostridial toxins are useful, forexample, as a means of delivering the therapeutic benefits of suchclostridial toxins to patients who are immunologically resistant to agiven clostridial toxin subtype, to patients who may have a lower thanaverage concentration of receptors to a given clostridial toxin heavychain binding domain, or to patients who may have a protease-resistantvariant of the membrane or vesicle toxin substrate (e.g., SNAP-25, VAMPand syntaxin). Hybrid and chimeric clostridial toxins are described inU.S. Pat. No. 8,071,110, which publication is hereby incorporated byreference in its entirety. Thus, in one embodiment, the engineeredclostridial toxin of the invention is an engineered hybrid clostridialtoxin, or an engineered chimeric clostridial toxin.

The term “clostridial toxin” is intended to embrace re-targetedclostridial toxins. In a re-targeted clostridial toxin, the clostridialtoxin is modified to include an exogenous ligand known as a TargetingMoiety (TM). The TM is selected to provide binding specificity for adesired target cell, and as part of the re-targeting process the nativebinding portion of the clostridial toxin (e.g. the H_(C) domain, or theH_(CC) domain) may be removed. Re-targeting technology is described, forexample, in: EP-B-0689459; WO 1994/021300; EP-B-0939818; U.S. Pat. No.6,461,617; U.S. Pat. No. 7,192,596; WO 1998/007864; EP-B-0826051; U.S.Pat. No. 5,989,545; U.S. Pat. No. 6,395,513; U.S. Pat. No. 6,962,703; WO1996/033273; EP-B-0996468; U.S. Pat. No. 7,052,702; WO 1999/017806;EP-B-1107794; U.S. Pat. No. 6,632,440; WO 2000/010598; WO 2001/21213; WO2006/059093; WO 2000/62814; WO 2000/04926; WO 1993/15766; WO 2000/61192;and WO 1999/58571; all of which are hereby incorporated by reference intheir entirety. Thus, in one embodiment, the engineered clostridialtoxin of the invention is an engineered re-targeted clostridial toxin.

The present invention also embraces clostridial toxins that have anon-native protease cleavage site. In such clostridial toxins, thenative protease cleavage site (also known as the activation site, asdescribed above) is modified or replaced with a protease cleavage sitethat is not native to that clostridial toxin (i.e. an exogenous cleavagesite). Such a site will require an exogenous protease for cleavage,which allows for improved control over the timing and location ofcleavage events. Non-native protease cleavage sites that may be employedin clostridial toxins include:

Enterokinase (DDDDK↓) Factor Xa (IEGR↓/IDGR↓) TEV(Tobacco Etch virus)(ENLYFQ↓G) Thrombin (LVPR↓GS) PreScission (LEVLFQ↓GP).

Additional protease cleavage sites include recognition sequences thatare cleaved by a non-cytotoxic protease, for example by the light chainof a clostridial neurotoxin. These include the SNARE (e.g. SNAP-25,syntaxin, VAMP) protein recognition sequences that are cleaved bynon-cytotoxic proteases such as the light chain of a clostridialneurotoxin. Clostridial toxins comprising non-native protease cleavagesites are described in U.S. Pat. No. 7,132,259, EP 1206554-B2 and US2007/0166332, all of which are hereby incorporated by reference in theirentirety. Also embraced by the term protease cleavage site is an intein,which is a self-cleaving sequence. The self-splicing reaction iscontrollable, for example by varying the concentration of reducing agentpresent.

The present invention also embraces clostridial toxins comprising a“destructive cleavage site”. In said clostridial toxins, a non-nativeprotease cleavage site is incorporated into the clostridial toxin, at alocation chosen such that cleavage at said site will decrease theactivity of, or inactivate, the clostridial toxin. The destructiveprotease cleavage site can be susceptible to cleavage by a localprotease, in the event that the clostridial toxin, followingadministration, migrates to a non-target location. Suitable non-nativeprotease cleavage sites include those described above. Clostridialtoxins comprising a destructive cleavage site are described in WO2010/094905 and WO 2002/044199, both of which are hereby incorporated byreference in their entirety.

The engineered clostridial toxins of the present invention, especiallythe light chain component thereof, may be PEGylated—this may help toincrease stability, for example duration of action of the light chaincomponent. PEGylation is particularly preferred when the light chaincomprises a BoNT/A, B or C₁ protease. PEGylation preferably includes theaddition of PEG to the N-terminus of the light chain component. By wayof example, the N-terminus of a light chain may be extended with one ormore amino acid (e.g. cysteine) residues, which may be the same ordifferent. One or more of said amino acid residues may have its own PEGmolecule attached (e.g. covalently attached) thereto. An example of thistechnology is described in WO2007/104567, which is hereby incorporatedby reference in its entirety.

The engineered clostridial toxins of the present invention may be freefrom the complexing proteins that are present in a naturally occurringclostridial toxin complex.

An engineered clostridial toxin of the present invention may alsocomprise a limited number of non-standard amino acids. Thus, in additionto the 20 standard amino acids, non-standard amino acids (such as4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovalineand α-methyl serine) may be substituted for amino acid residues of theengineered clostridial toxins of the present invention. A limited numberof non-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted forclostridial polypeptide amino acid residues. The engineered clostridialtoxins of the present invention can also comprise non-naturallyoccurring amino acid residues.

Non-naturally occurring amino acids include, without limitation,trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline,trans-4-hydroxy-proline, N-methylglycine, allo-threonine,methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine,nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline,2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and4-fluorophenylalanine. Several methods are known in the art forincorporating non-naturally occurring amino acid residues into proteins.For example, an in vitro system can be employed wherein nonsensemutations are suppressed using chemically aminoacylated suppressortRNAs. Methods for synthesizing amino acids and aminoacylating tRNA areknown in the art. Transcription and translation of plasmids containingnonsense mutations is carried out in a cell free system comprising an E.coli S30 extract and commercially available enzymes and other reagents.Proteins are purified by chromatography. See, for example, Robertson etal., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al.,Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,translation is carried out in Xenopus oocytes by microinjection ofmutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti etal., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. colicells are cultured in the absence of a natural amino acid that is to bereplaced (e.g., phenylalanine) and in the presence of the desirednon-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Thenon-naturally occurring amino acid is incorporated into the polypeptidein place of its natural counterpart. See, Koide et al., Biochem.33:7470-6, 1994.

The engineered clostridial toxins of the present invention can beproduced using recombinant nucleic acid technologies. Thus, in oneembodiment, an engineered clostridial toxin (as described above) is arecombinant engineered clostridial toxin.

In another aspect, the present invention provides a nucleic acid (forexample, a DNA) comprising a nucleic acid sequence encoding anengineered clostridial toxin as described above. In one embodiment, thenucleic acid sequence is prepared as part of a DNA vector comprising apromoter and a terminator.

In a preferred embodiment, the vector has a promoter selected from:

Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lacoperator IPTG 0.2 mM (0.05-2.0 mM)

In another preferred embodiment, the vector has a promoter selectedfrom:

Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lacoperator IPTG 0.2 mM (0.05-2.0 mM) T5-lac operator IPTG 0.2 mM (0.05-2.0mM)

The nucleic acid molecules of the invention may be made using anysuitable process known in the art. Thus, the nucleic acid molecules maybe made using chemical synthesis techniques. Alternatively, the nucleicacid molecules of the invention may be made using molecular biologytechniques.

The DNA construct of the present invention is preferably designed insilico, and then synthesised by conventional DNA synthesis techniques.

The above-mentioned nucleic acid sequence information is optionallymodified for codon-biasing according to the ultimate host cell (e.g. E.coli) expression system that is to be employed.

In one embodiment, the nucleic acid sequence encoding an engineeredclostridial toxin as described above is a nucleic acid sequence havingat least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98 or 99%)sequence identity to a nucleic acid sequence selected from SEQ ID NOs:2, 4, and 6. In one embodiment, the nucleic acid sequence has at least90% sequence identity to a nucleic acid sequence selected from SEQ IDNOs: 2, 4, and 6.

The present invention also provides polypeptides encoded by nucleic acidsequences as described above. Thus, in one aspect, the present inventionprovides a polypeptide comprising an amino acid sequence having at least70% (for example, at least 75, 80, 85, 90, 95, 97, 98 or 99%) sequenceidentity to an amino acid sequence selected from SEQ ID NOs: 1, 3 and 5.In one embodiment, the amino acid sequence has at least 90% sequenceidentity to an amino acid sequence selected from SEQ ID NOs: 1, 3 and 5.

In one embodiment, the engineered clostridial toxin of the invention isan engineered BoNT/A as described above, and said engineered BoNT/Acomprises (or consists of) an amino acid sequence having at least 70%(for example, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%)sequence identity to an amino acid sequence selected from SEQ ID NOs: 1,3 and 5.

In one embodiment, the engineered clostridial toxin of the invention isan engineered BoNT/A as described above, and said engineered BoNT/Acomprises (or consists of) the amino acid sequence of SEQ ID NO: 1, 3 or5.

In one aspect, the invention provides a polypeptide comprising (orconsisting of) the amino acid sequence of SEQ ID NO: 1, 3 or 5.

In one aspect, the invention provides a nucleic acid encoding anengineered clostridial toxin as described above, wherein said nucleicacid comprises a nucleic acid sequence having at least 70% (for example,at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequenceidentity to a nucleic acid sequence selected from SEQ ID NOs: 2, 4 and6. In one embodiment, the nucleic acid comprises (or consists of) thenucleic acid sequence of SEQ ID NO: 2, 4 or 6.

In one aspect, the invention provides a nucleic acid comprising (orconsisting of) the nucleic acid sequence of SEQ ID NO: 2, 4 or 6.

In one embodiment, the engineered clostridial toxin of the invention isan engineered BoNT/E as described above, and said engineered BoNT/Ecomprises an amino acid sequence having at least 70% (for example, atleast 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequence identityto SEQ ID NO: 7.

In one aspect, the invention provides a nucleic acid encoding anengineered clostridial toxin as described above, wherein said nucleicacid comprises a nucleic acid sequence having at least 70% (for example,at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequenceidentity to SEQ ID NO: 8.

The “percent sequence identity” between two or more nucleic acid oramino acid sequences is a function of the number of identical positionsshared by the sequences. Thus, % identity may be calculated as thenumber of identical nucleotides/amino acids divided by the total numberof nucleotides/amino acids, multiplied by 100. Calculations of %sequence identity may also take into account the number of gaps, and thelength of each gap that needs to be introduced to optimize alignment oftwo or more sequences. Sequence comparisons and the determination ofpercent identity between two or more sequences can be carried out usingspecific mathematical algorithms, such as BLAST, which will be familiarto a skilled person.

In one aspect, the present invention provides a method of producing asingle-chain engineered clostridial toxin protein having a light chainand a heavy chain, the method comprising expressing a nucleic acid (saidnucleic acid being as described above) in a suitable host cell, lysingthe host cell to provide a host cell homogenate containing thesingle-chain engineered clostridial toxin protein, and isolating thesingle-chain engineered clostridial toxin protein.

In another aspect, the present invention provides a method of activatingan engineered clostridial toxin, the method comprising providing asingle-chain engineered clostridial toxin protein obtainable by themethod of producing a single-chain engineered clostridial toxin proteinas described above, contacting the polypeptide with a protease thatcleaves the polypeptide at a recognition site (cleavage site) locatedbetween the light chain and heavy chain, thereby converting thepolypeptide into a di-chain polypeptide wherein the light chain andheavy chain are joined together by a disulphide bond.

The engineered clostridial toxins of the invention may be used toprevent or treat certain medical or cosmetic diseases and conditions.Thus, in a further aspect, the present invention provides an engineeredclostridial toxin as described above, for use in medicine.

In a related aspect, the present invention provides an engineeredclostridial toxin as described above, for use in the prevention ortreatment of a disease or condition selected from: strabismus,blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibulardystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limbdystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis),beauty therapy (cosmetic) applications benefiting from cell/muscleincapacitation (via SNARE down-regulation or inactivation),neuromuscular disorder or condition of ocular motility (e.g. concomitantstrabismus, vertical strabismus, lateral rectus palsy, nystagmus,dysthyroid myopathy), writer's cramp, blepharospasm, bruxism, Wilson'sdisease, tremor, tics, segmental myoclonus, spasms, spasticity due tochronic multiple sclerosis, spasticity resulting in abnormal bladdercontrol, animus, back spasm, charley horse, tension headaches, levatorpelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease,stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focalspasticity, spasmodic colitis, neurogenic bladder, anismus, limbspasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia,lacrimation, hyperhydrosis, excessive salivation, excessivegastrointestinal secretions, muscle pain (e.g. pain from muscle spasms),headache pain (e.g. tension headache), brow furrows, skin wrinkles,cancer, uterine disorders, uro-genital disorders,urogenital-neurological disorders, chronic neurogenic inflammation, anda smooth muscle disorder.

In use, the present invention employs a pharmaceutical composition,comprising an engineered clostridial toxin, together with at least onecomponent selected from a pharmaceutically acceptable carrier,excipient, adjuvant, propellant and/or salt.

The engineered clostridial toxins of the present invention may beformulated for oral, parenteral, continuous infusion, inhalation ortopical application. Compositions suitable for injection may be in theform of solutions, suspensions or emulsions, or dry powders which aredissolved or suspended in a suitable vehicle prior to use.

In the case of an engineered clostridial toxin that is to be deliveredlocally, the engineered clostridial toxin may be formulated as a cream(e.g. for topical application), or for sub-dermal injection.

Local delivery means may include an aerosol, or other spray (e.g. anebuliser). In this regard, an aerosol formulation of an engineeredclostridial toxin enables delivery to the lungs and/or other nasaland/or bronchial or airway passages.

Engineered clostridial toxins of the invention may be administered to apatient by intrathecal or epidural injection in the spinal column at thelevel of the spinal segment involved in the innervation of an affectedorgan.

A preferred route of administration is via laparoscopic and/orlocalised, particularly intramuscular, injection.

The dosage ranges for administration of the engineered clostridialtoxins of the present invention are those to produce the desiredtherapeutic effect. It will be appreciated that the dosage rangerequired depends on the precise nature of the engineered clostridialtoxin or composition, the route of administration, the nature of theformulation, the age of the patient, the nature, extent or severity ofthe patient's condition, contraindications, if any, and the judgement ofthe attending physician. Variations in these dosage levels can beadjusted using standard empirical routines for optimisation.

Suitable daily dosages (per kg weight of patient) are in the range0.0001-1 ng/kg, preferably 0.0001-0.5 ng/kg, more preferably 0.002-0.5ng/kg, and particularly preferably 0.004-0.5 ng/kg. The unit dosage canvary from less than 1 picogram to 30 ng, but typically will be in theregion of 0.01 to 1 ng per dose, which may be administered daily orpreferably less frequently, such as weekly or six monthly. Aparticularly preferred dosing regimen is based on 0.05 ng of engineeredclostridial toxin as the 1× dose. In this regard, preferred dosages arein the range 1×-100× (i.e. 0.05-5 ng).

Fluid dosage forms are typically prepared utilising the engineeredclostridial toxin and a pyrogen-free sterile vehicle. The engineeredclostridial toxin, depending on the vehicle and concentration used, canbe either dissolved or suspended in the vehicle. In preparing solutionsthe engineered clostridial toxin can be dissolved in the vehicle, thesolution being made isotonic if necessary by addition of sodium chlorideand sterilised by filtration through a sterile filter using aseptictechniques before filling into suitable sterile vials or ampoules andsealing. Alternatively, if solution stability is adequate, the solutionin its sealed containers may be sterilised by autoclaving.Advantageously additives such as buffering, solubilising, stabilising,preservative or bactericidal, suspending or emulsifying agents and orlocal anaesthetic agents may be dissolved in the vehicle.

Dry powders, which are dissolved or suspended in a suitable vehicleprior to use, may be prepared by filling pre-sterilised ingredients intoa sterile container using aseptic technique in a sterile area.Alternatively the ingredients may be dissolved into suitable containersusing aseptic technique in a sterile area. The product is then freezedried and the containers are sealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous orintradermal injection, are prepared in substantially the same manner,except that the sterile components are suspended in the sterile vehicle,instead of being dissolved and sterilisation cannot be accomplished byfiltration. The components may be isolated in a sterile state oralternatively it may be sterilised after isolation, e.g. by gammairradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone isincluded in the composition(s) to facilitate uniform distribution of thecomponents.

Administration in accordance with the present invention may takeadvantage of a variety of delivery technologies including microparticleencapsulation, viral delivery systems or high-pressure aerosolimpingement.

FIGURE LEGENDS

FIG. 1.

SDS-PAGE purification of CatH_(N) _(_)v1 (FIG. 1A), CatH_(N) _(_)v2(FIG. 1B), and CatH_(N) _(_)v3 (FIG. 1C) purification.

FIG. 2.

Percentage SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN)for CatH_(N) _(_)v1. Rat embryonic spinal cord neurons were cultured forthree weeks and treated with CatH_(N) _(_)v1 for 24 h, before Westernblotting with SNAP-25 specific antibody. Data is mean±SEM from threeindependent experiments in triplicate.

FIG. 3.

The potency (t₅₀) of BoNT/A and CatH_(N) _(_)v1 in the mouse phrenicnerve hemi-diaphragm assay (mPNHD). Data points are individualhemi-diaphragm preparations and means±SEM. CatH_(N) _(_)v1 wasstatistically significantly slower than reference protein BoNT/A (ListBiological Laboratories). 1-way ANOVA and Dunnett's multiple comparisonstest. ** p<0.01, *** p<0.001, **** p<0.0001 (1-way ANOVA and Dunnett'smultiple comparisons test).

FIG. 4.

Percentage SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN)for CatH_(N) _(_)v2. Rat embryonic spinal cord neurons were cultured forthree weeks and treated with CatH_(N) _(_)v2 for 24 h, before Westernblotting with SNAP-25 specific antibody. Data is mean±SEM from threeindependent experiments in triplicate.

FIG. 5.

The potency (t₅₀) of BoNT/A and CatH_(N) _(_)v2 in the mouse phrenicnerve hemi-diaphragm assay (mPNHD). Data points are individualhemi-diaphragm preparations and means±SEM. CatH_(N) _(_)v2 isstatistically equivalent to the reference protein BoNT/A (ListBiological Laboratories). 1-way ANOVA and Dunnett's multiple comparisonstest. ** p<0.01, *** p<0.001, **** p<0.0001 (1-way ANOVA and Dunnett'smultiple comparisons test).

FIG. 6.

Percentage SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN)for CatH_(N) _(_)v3. Rat embryonic spinal cord neurons were cultured forthree weeks and treated with CatH_(N) _(_)v3 for 24 h, before Westernblotting with SNAP-25 specific antibody. Data is mean±SEM from threeindependent experiments in triplicate.

FIG. 7.

The potency (t₅₀) of BoNT/A and CatH_(N) _(_)v3 in the mouse phrenicnerve hemi-diaphragm assay (mPNHD). Data points are individualhemi-diaphragm preparations and means±SEM. CatH_(N) _(_)v3 wasstatistically significantly slower than the reference protein BoNT/A(List Biological Laboratories). 1-way ANOVA and Dunnett's multiplecomparisons test. ** p<0.01, *** p<0.001, **** p<0.0001 (1-way ANOVA andDunnett's multiple comparisons test).

FIG. 8.

Isoelectric focusing analysis. All three CatH_(N) constructs possess anincreased observed p/compared to unmodified BoNT/A.

FIG. 9.

SDS-PAGE purification of CatLC construct.

FIG. 10.

Catalytic activity of CatLC compared to BoNT/E LC reference, with pEC₅₀values obtained in the BoTest A/E BoNT Detection Kit (BioSentinalCat#A1004), following manufacturer's instructions. Data showsmean±standard deviation from one independent experiment in triplicate.

SEQUENCES

-   SEQ ID NO: 1. Engineered BoNT/A, “CatH_(N) _(_)v1”, amino acid    sequence.-   SEQ ID NO: 2. Engineered BoNT/A, “CatH_(N) _(_)v1”, nucleic acid    sequence.-   SEQ ID NO: 3. Engineered BoNT/A, “CatH_(N) _(_)v2”, amino acid    sequence.-   SEQ ID NO: 4. Engineered BoNT/A, “CatH_(N) _(_)v2”, nucleic acid    sequence.-   SEQ ID NO: 5. Engineered BoNT/A, “CatH_(N) _(_)v3”, amino acid    sequence.-   SEQ ID NO: 6. Engineered BoNT/A, “CatH_(N) _(_)v3”, nucleic acid    sequence.-   SEQ ID NO: 7. Engineered BoNT/E light chain, “CatLC”, amino acid    sequence.-   SEQ ID NO: 8. Engineered BoNT/E light chain, “CatLC”, nucleic acid    sequence.

SEQ ID NO: 1. Engineered BoNT/A, ″CatH_(N)_v1″, amino acid sequence. MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKRRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMRYKRRFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSRDRPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLSEQ ID NO: 2. Engineered BoNT/A, ″CatH_(N)_v1″, nucleic acid sequence. ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAcgtCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCgtTACAAAcgtcgTTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCcgtGACcgtCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGACGTCCGCTGSEQ ID NO: 3. Engineered BoNT/A, ″CatH_(N)_v2″, amino acid sequence. MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLKKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATKAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIAKKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNKIKFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLKGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLSEQ ID NO: 4. Engineered BoNT/A, ″CatH_(N)_v2″, nucleic acid sequence. ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAAgAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTaAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAAgAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAAgATTAAgTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGAagGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTGSEQ ID NO: 5. Engineered BoNT/A, ″CatH_(N)_v3″, amino acid sequence. MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLKPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPKLGTFALVSYKANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYKEKEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLSEQ ID NO: 6. Engineered BoNT/A, ″CatH_(N)_v3″, nucleic acid sequence. ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCgAtACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAAaCCGAGCCGTGTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTTCATTCCGGAGATTGCGATCCCGaaGTTGGGTACCTTCGCGCTGGTGTCCTACAagGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATCAACTACCAATACAACCAGTACAaGGAAaAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTdTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGACGTCCGCTGSEQ ID NO: 7. Engineered BoNT/E light chain,″CatLC″, amino acid sequence. MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSMPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNKFHIGDASAVEIKFSKGSQHILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSINEFIQDPALTLMHELIHSLHGLYGAKGITTTCIITQQKNPLITNRKGINIEEFLTFGGNDLNIITVAQYNDIYTNLLNDYRKIASKLSKVQVSNPQLNPYKDIFQEKYGLDKDASGIYSVNINKFDDILKKLYSFTEFDLATKFQVKCRETYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIIKPITGRGLVKK IIRFAVDKLAAALEHHHHHHSEQ ID NO: 8. Engineered BoNT/E light chain,″CatLC″, amino acid sequence. ATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAAGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTCGGGATCGAGGGAAGGATTTCAGAATTCGGATCCATGCCAAAAATCAACAGCTTTAATTACAATGACCCTGTAAACGATCGTACCATCCTATACATAAAGCCGGGTGGGTGTCAAGAGTTCTACAAATCTTTCAATATTATGAAGAATATATGGATTATACCTGAGCGTAACGTTATTGGTACGACACCGCAAGATTTTCATCCACCTACTTCGTTGAAGAACGGTGACTCTTCCTATTACGACCCCAATTATCTCCAGTCGGATGAAGAGAAGGACAGATTCCTTAAAATAGTAACCAAAATCTTTAACAGGATTAATAACAATCTATCCGGAGGTATTTTGCTTGAAGAGCTTAGTAAAGCTAATCCTTACCTAGGTAACGATAATACACCAGACAACAAGTTTCATATAGGCGATGCATCCGCCGTGGAAATCAAATTTAGCAAGGGATCACAGCATATTCTCTTGCCCAACGTTATTATAATGGGGGCGGAACCAGATTTATTTGAAACAAATTCGAGTAATATTAGCCTGAGAAATAACTATATGCCGTCAAACCATGGGTTCGGTAGCATAGCGATCGTTACTTTTTCTCCCGAATACAGTTTTCGCTTCAATGATAATAGTATAAATGAGTTTATCCAAGACCCCGCACTCACGCTTATGCACGAACTCATACACTCTTTACACGGCCTGTATGGCGCTAAGGGGATAACCACTACGTGTATCATTACTCAGCAAAAGAACCCATTGATAACGAACAGGAAGGGCATTAACATCGAGGAATTTCTTACATTTGGAGGCAACGATCTGAACATTATAACTGTCGCACAGTACAATGACATCTATACCAACTTACTAAATGATTATAGAAAAATCGCTTCTAAGTTATCCAAGGTTCAAGTCTCAAACCCTCAACTGAATCCGTATAAGGACATATTCCAAGAGAAATATGGATTAGACAAAGACGCGTCAGGAATCTATTCGGTAAACATTAACAAATTCGACGATATTTTGAAGAAACTTTACAGCTTCACGGAGTTCGACTTGGCCACCAAATTCCAGGTCAAATGCCGAGAGACATACATCGGACAGTATAAGTATTTCAAGCTGTCGAATCTCCTGAATGATTCCATATACAACATTAGTGAGGGTTACAATATAAATAACCTAAAGGTGAATTTCCGAGGCCAAAACGCCAACCTAAATCCGCGCATCATTAAACCCATCACAGGACGGGGGTTAGTGAAGAAAATAATCCGGTTTGCGGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCAC CACCACCAC

EXAMPLES

The following Examples serve to illustrate particular embodiments of theinvention, and do not limit the scope of the invention defined in theclaims in any way.

Example 1 Identification of Preferred Clostridial Toxin Amino Acids forModification.

The amino acids identified as suitable candidates for modification(mutation sites) were selected using a number of different criteria.

1. Location of residue within BoNT molecule (within H_(N), excludingbelt region)2. Location with regard to secondary/tertiary structure;3. Type of residue;4. Degree of surface exposure.

Acidic, neutral, polar and hydrophobic residues were considered forselection.

Exposed residues were determined using AreaIMol from the CCP4 suite.Each structure was analysed by AreaIMol, and exposed residues wereidentified as having a sum value greater than 55.

Secondary structures within the H_(N) of each Subtype and TeNT wereidentified using a secondary structure assignment program (Stride WebInterface). Regions assigned as forming α-helix, β-strand or 3₁₀-helixwere excluded from the selection.

Sequences Used

Accession numbers:

BoNT/A: P10845 BoNT/B: P10844 BoNT/C: P18640 BoNT/D: P19321 BoNT/E:Q00496 BoNT/F: YP_001390123 BoNT/G: Q60393

Structural Data Source

Crystal structures of BoNT/A (3BTA.pdb), BoNT/B (1EPW), and BoNT/E(3FFZ.pdb) obtained from RCSB.

Homology modelling of BoNT/C₁, BoNT/D, BoNT/F, and BoNT/G performedusing LOOPP and the following sequences, respectively: P18640, P19321,YP_001390123, and Q60393.

Preferred clostridial toxin amino acid residues for modification in theclostridial toxin H_(N) domain:

BoNT/A:

D474, N476, D484, N486, I487, E488, A489, A490, E491, D546, E558, E560,H561, I566, L568, N570, S571, L577, N578, A597, E599, A601, E620, V621,T623, D625, T631, N645, L647, D650, D651, I668, E670, A672, V675, S683,I685, A686, N687, N752, Q753, T755, E756, E757, E758, N760, N761, I762,N763, D825, I831, G832, T847, D848, and D858

BoNT/B:

V443, G444, D453, S468, D533, E534, N535, T545, L548, D549, I550, D552,S557, L564, S566, N582, V584, N609, L619, N632, E633, G637, A646, I655,E657, V662, E669, S670, I672, D673, N739, I740, N748, N750, I818, G819,T834, I842, N845, and S858

BoNT/C₁:

L451, D452, C453, E455, V472, T474, D475, L478, N483, E484, E485, E487,I489, L555, S556, D557, N558, E560, D561, E569, N574, S575, T584, G592,Q594, G596, D617, N640, S641, V642, G645, N646, E661, E665, T667, A670,S678, V680, Q681, E682, S750, G751, S759, Q760, V826, G827, N842, T843,N847, and N853

BoNT/D:

Q469, E470, E473, N474, D479, E480, N482, V483, Q484, N485, S487, D488,S552, N553, N554, V555, E556, N557, I558, L560, T562, S563, V564, G569,S571, N572, G588, Q590, T614, D616, S619, S622, N636, S637, L639, G641,N642, E657, E661, T663, A666, V669, S674, I676, Q677, E678, S746, G747,D749, E751, N752, I753, Q756, N818, V822, G823, E837, N838, T839, N843,N849, and N850

BoNT/E:

D474, N476, E479, E480, D484, N486, I487, E488, A489, A490, E491, E492,L496, D497, Q500, Q501, L504, N507, D509, N510, N514, S516, E518, Q527,L530, N533, I534, E535, N539, Y548, I566, L568, D589, A597, E599, A601,L604, Y612, E620, N645, L647, Y648, D651, E737, E741, Y803, Y824, D825,G828, I831, G832, and D835

BoNT/F:

N463, E464, N468, T469, D474, D475, T476, T477, N478, N482, N485, N495,I499, Q501, I502, Q505, T506, N508, T509, V511, D513, D521, S522, S526,E527, I528, E529, V534, D535, L536, E549, G550, T552, N553, S558, E566,E567, S568, V586, H587, Q608, D613, A616, D617, S619, N630, N633, N639,E654, V656, E658, L660, T663, L665, V666, S671, I673, G674, S675, S676,E677, N678, T746, N751, L753, E754, E756, N758, I759, N760, N761, S799,S821, I822, N840, S841, E845, L846, S847, S848, T850, N851, D852, I854,L855, and 1856

BoNT/G:

N480, Q482, N483, N484, T485, E487, D540, N562, N570, N571, N572, T588,V589, T615, D621, N637, E638, E642, N643, I660, E662, I667, E674, S675,V677, G678, N679, S747, N755, D757, L823, D839, I841, D844, S846, andL847

TeNT:

A457, S458, L459, D461, L462, E486, E487, Q490, D491, N497, N504, D557,T571, T572, L573, Q574, N580, S581, N588, S589, T590, S598, Q605, G606,Q608, T631, I633, S640, Q655, E658, G659, N660, E675, I677, E679, T681,V684, A691, E692, S694, T695, Q696, A772, D773, E774, S862, N866, L867and D868

Preferred clostridial toxin amino acid residues for modification in theBoNT/E light chain:

N5, N8, N10, D11, N14, D15, Q27, E28, N72, Q75, N118, D121, N122, Q123,N138, Q237, Q290, N297, N362, N365, D366, N378, and N379. Example 2Design of Engineered BoNT/a Molecules

Three different examples of an engineered BoNT/A molecule according tothe present invention were produced.

Using the method described in Example 1, a total of 55 residues wereidentified as candidates for mutation in the BoNT/A H_(N) domain. Thesuitability of the residues was further assessed by visual inspection ofthe BoNT/A crystal structure to give a list of 11 preferred candidates(N476, N763, N687, E599, I831, N761, N578, V675, I685, T755, E757). Afurther six residues were chosen based on functional data that showedthat these residues were amenable to mutation without adverselyaffecting protein function. Four of these residues were within thecandidate list (L647, D650, D651, T847) and two were not (S564, I849).With the 11 residues from the candidate list plus the six fromfunctional data, a total of 17 residues were selected for mutation.

From the 17 residues selected, 3 constructs were made: CatH_(N) _(_)v1,CatH_(N) _(_)v2 and CatH_(N) _(_)v3. The mutations for the CatH_(N)constructs are shown in Table 3 below:

TABLE 3 CatH_(N) constructs with mutations listed and calculated pI.Number of Calculated Calculated ΔpI relative Mutations Mutations pI toBoNT/A (pI 6.4) CatHN_v1 S564R, 6 7.4 1.0 L647R, D650R, D651R, T847R,I849R CatHN_v2 N476K, 6 7.3 0.9 N763K, N687K, E599K, I831K, N761KCatHN_v3 N578K, 5 7.1 0.7 V675K, I685K, T755K, E757K [ΔpI = change inisoelectric point]

Purification of CatH_(N) _(_)v1, CatH_(N) _(_)v2 and CatH_(N) _(_)v3 isshown in FIGS. 1A, 1B and 1C, respectively.

Example 3 Cloning, Expression and Purification

DNA constructs encoding the engineered BoNT/A molecules described inExample 2 were synthesised, cloned into the pJ401 expression vector andthen transformed into BL21 (DE3) E. coli. This allowed for solubleover-expression of the recombinant engineered BoNT/A molecules in E.coli.

The recombinant engineered BoNTs were purified using classicalchromatography techniques from the E. coli lysates. An initialpurification step using a cation-exchange resin was employed, followedby an intermediate purification step using a hydrophobic interactionresin. The recombinant engineered BoNT single-chain was then cleaved byproteolysis, resulting in the activated di-chain engineered BoNT. Afinal purification step was then employed to remove remainingcontaminants.

Example 4

Characterization of purified engineered BoNTs

The engineered BoNTs described in Example 2 above were characterisedexperimentally.

The ability of the engineered BoNTs to enter neurons and cleave SNAP-25(the target of BoNT/A) was assessed using rat embryonic spinal cordneurons (eSCN). Potency of the engineered BoNTs was further assessedusing the mouse phrenic nerve hemi-diaphragm assay (mPNHD).

CatH_(N) _(_)v1:

The first set of mutations added were substitutions to Arginine:

S564R, L647R, D650R, D651R, T847R, I849R

The CatH_(N) _(_)v1 molecule was tested in the rat embryonic spinal cordneuron (eSCN) SNAP-25 cleavage assay, and found to be equipotent potentto BoNT/A (BoNT/A) (FIG. 2).

A positive result was also demonstrated in the mouse phrenic nervehemi-diaphragm (mPNHD) assay (FIG. 3).

CatH_(N) _(_)v2:

The second set of mutations were substitutions to Lysine:

N476K, N763K, N687K, E599K, I831K, N761K

The CatH_(N) _(_)v2 protein was tested in the eSCN SNAP-25 cleavageassay, and found to retain the ability to enter the cells and cleaveSNAP-25. In the mPNHD assay CatH_(N) _(_)v2 was equipotent to BoNT/A(FIGS. 4 and 5).

CatH_(N) _(_)v3:

The third set of mutations were substitutions to Lysine:

N578K, V675K, 1685K, T755K, E757K

The CatH_(N) _(_)v3 molecule was tested in the eSCN SNAP-25 cleavageassay, and found to retain the ability to enter the cells and cleaveSNAP-25. Similarly, a positive result was also demonstrated in the mPNHDassay (FIGS. 6 and 7).

Isoelectric Focusing

All three CatH_(N) constructs possess an increased p/compared tounmodified BoNT/A (FIG. 8).

Example 5 Modifications in the Light Chain of BoNT/E

Due to the modularity of the botulinum toxin, a BoNT/E light chainconstruct with an N-terminal maltose binding protein (MBP) tag and aC-terminal 6 histidine tag (6HT) was used as a surrogate to assay BoNT/Eactivity when mutated and characterised.

A BoNT/E light chain construct (“CatLC”) was prepared having themutations shown in table below.

TABLE 4 Construct with mutations listed, calculated pI and calculatedΔpI. Number of Calculated Calculated ΔpI relative to Mutations MutationspI MBP-LC/E (pI 6.3) CatLC Q123K 3 6.6 0.3 N138K Q237K

The construct was expressed in BL21 DE3 cells and purified usingaffinity chromatography. Purification is shown in FIG. 9.

Assessment of the construct for catalytic activity showed that themodified light chain retained the catalytic activity of unmodified,light chain (FIG. 10).

1. An engineered clostridial toxin comprising an amino acid modification that increases the isoelectric point of the toxin to a value that is at least 0.2 pI units higher than the isoelectric point of an otherwise identical toxin lacking the modification, wherein the modification is not located in the clostridial toxin binding domain.
 2. The toxin of claim 1, wherein the modification is located in the clostridial toxin translocation domain.
 3. The toxin of claim 1, wherein the modification is located in the clostridial toxin light chain.
 4. The toxin of claim 3, wherein the modification does not introduce an E3 ligase recognition motif into the clostridial toxin light chain.
 5. The toxin of claim 1, wherein the modification increases the isoelectric point of the toxin to a value that is at least 0.5 pI units higher than the isoelectric point of an otherwise identical clostridial toxin lacking the modification. 6-7. (canceled)
 8. The toxin of claim 1, wherein the modification increases the isoelectric point of the toxin to a value that is between 2 and 5 pI units higher than the isoelectric point of an otherwise identical clostridial toxin lacking the modification.
 9. The toxin of claim 1, having an isoelectric point of at least 6.5.
 10. (canceled)
 11. The toxin of claim 1 having an isoelectric point of between 6.5 and 7.5.
 12. The toxin of claim 1, wherein the modification is an amino acid substitution, an amino acid insertion, or an amino acid deletion.
 13. The toxin of claim 12, wherein the modification is a substitution of an acidic amino acid residue with a basic amino acid residue, a substitution of an acidic amino acid residue with an uncharged amino acid residue, or a substitution of an uncharged amino acid residue with a basic amino acid residue.
 14. The toxin of claim 1, comprising from 1 to 90 amino acid modifications.
 15. The toxin of claim 1, comprising at least three amino acid modifications.
 16. The toxin of claim 1, comprising from 4 to 40 amino acid modifications.
 17. The toxin of claim 1, wherein the modification is to a surface exposed amino acid residue.
 18. The toxin of claim 1, wherein the modification is to an amino acid residue selected from: aspartic acid, glutamic acid, histidine, asparagine, glutamine, serine, threonine, alanine, glycine, valine, leucine, and isoleucine.
 19. The toxin of claim 18, wherein the amino acid residue is substituted with lysine or arginine.
 20. A nucleic acid comprising a nucleic acid sequence encoding the toxin of claim
 1. 21. A method of producing a single-chain engineered clostridial toxin protein having a light chain and a heavy chain, the method comprising expressing the nucleic acid of claim 20 in a suitable host cell, lysing the host cell to provide a host cell homogenate containing the single-chain engineered clostridial toxin protein, and isolating the single-chain engineered clostridial toxin protein.
 22. A method of activating an engineered clostridial toxin, the method comprising providing a single-chain engineered clostridial toxin protein obtainable by the method of claim 21, contacting the protein with a protease that cleaves the protein at a recognition site located between the light chain and heavy chain, and converting the protein into a di-chain polypeptide wherein the light chain and heavy chain are joined together by a disulphide bond.
 23. (canceled)
 24. A method for treating or preventing a disease or condition comprising administering the toxin of claim 1 to a patient in need thereof, the disease or condition being selected from: strabismus, blepharospasm, squint, dystonia, torticollis, a cosmetic condition that is treated by cell or muscle incapacitation, writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, charley horse, tension headaches, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain, headache pain, brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, chronic neurogenic inflammation, and a smooth muscle disorder.
 25. (canceled) 